Methods and reagents for the treatment of multiple sclerosis

ABSTRACT

The invention features methods and reagents for the diagnosis, monitoring, and treatment of multiple sclerosis. The invention is based in part on the discovery that Chlamydia is present in patients with multiple sclerosis, and that anti-chlamydial agents improve or sustain neurological function in these patients.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. Ser. No.09/528,348, filed Mar. 17, 2000 (now allowed), which is acontinuation-in-part of U.S. Ser. No. 09/073,661, filed May 6, 1998 (nowallowed), which is a continuation-in-part of U.S. Ser. No. 09/025,174,filed Feb. 18, 1998 (now allowed), which is a continuation-in-part ofU.S. Ser. No. 08/911,593, filed Aug. 14, 1997 (now abandoned). U.S. Ser.No. 09/528,348 also claims benefit of the filing dates of U.S.Provisional Application No. 60/125,598, filed Mar. 19, 1999, and U.S.Provisional Application Nos. 60/176,662, 60/176,940, and 60/176,784,each filed Jan. 18, 2000.

BACKGROUND OF THE INVENTION

[0002] Multiple Sclerosis (MS) is a chronic inflammatory disease of thecentral nervous system (CNS) in which the predominant pathologicfindings are demyelination accompanied by disruption of underlying axons(Trapp et al., New Engl. J. Med. 338:278-285, 1998; Prineas, J. W.,“Pathology of Multiple Sclerosis” in: Cook S D, ed. Handbook of MultipleSclerosis. New York: Marcel Dekker, Inc, 1990:187-215). The diseaseaffects young adults who usually present with a relapsing, remittingpattern of neurologic involvement and progress to a chronic phase withincreasing difficulty in ambulation and coordination. The etiology of MSis not known, but there is considerable indirect evidence that arguesfor the role of an infectious agent(s) in the pathogenesis of thedisease. Epidemiological studies strongly suggest that a CNS infectionin early childhood is a key factor in the development of MS (Kurtzke,Clin. Microbiol. Rev. 6:382-427, 1993). Viral infections have long beenthought to play a possible role in the pathogenesis of MS becauseviruses are known to cause demyelinating disease in experimentalanimals, often present clinically with relapsing, remitting symptoms,and can cause disease with long periods of latency (Cook and Dowling,Neurology 30:80-91, 1980; Johnson, R. T.,. Viral infections of theNervous System. New York: Raven Press, 1982). Studies to date, however,have failed to identify any virus as playing a major role in MS,although activated human herpes virus 6 (HHV-6) has been identifiedrecently in brains of MS patients (Sanders et al., J. Neurovirol.2:249-258, 1996; Challoner et al., Proc. Natl. Acad. Sci. USA92:7440-7444, 1995; Merelli, J. Neurol. 244:450-454, 1997). Although animmune response to this virus is seen during acute exacerbations, therole of HHV-6 infection in MS remains unclear (Soldan et al., NatureMed. 3:1394-1397, 1997).

[0003] Current opinion thus favors MS to be an autoimmune diseasedirected against self neural antigens (Martin et al., Annu. Rev.Immunol. 10:153-169, 1992). To reconcile the role of environment in thepathogenesis of MS as well as the absence of an identifiable infectiouspathogen, it is believed that infectious agents may act to trigger anautoimmune process. Such an autoimmune response may result fromstructural similarities between an infectious agent and neural antigens(antigenic mimicry) or from an expansion of self autoreactive T cellclones in response to bacterial or viral superantigens (Brocke et al.,Nature 65:642-646, 1993; Jahnke et al., Science 229:282-284, 1985;Marrack and Kappler, Science 248:325-329, 1998; Oldstone, J. Autoimmun.2(S):187-194, 1989). Evidence that MS is a disease mediated by T cellsthat recognize neural antigens has been hard to justify, since measuresdirected at either eliminating or reducing helper T cell function havenot changed the natural history of MS (Sriram and Rodriguez, Neurology48:469-473, 1997). Improved methods of diagnosing MS would facilitateidentification of treatable pathogens and expedite commencement oftreatment.

[0004] Over the last few years, therapy with β-IFN has emerged as ameans of reducing the morbidity of MS. Both β-IFNs (β-1a and β-1b)reduce the number of clinical relapses and slow the progression of thedisease. In addition, magnetic resonance imaging (MRI) studiesdemonstrate a decrease in the number of new inflammatory cerebrallesions in patients receiving β-IFN. Although β-IFN was introduced as atherapeutic agent for MS based on its anti-viral properties, the reasonsfor the therapeutic benefit of β-IFN for MS remain unclear. Thus far, noviral agent has been consistently found to be associated with MS.

SUMMARY OF THE INVENTION

[0005] In a first aspect, the present invention features a method ofdiagnosing or monitoring multiple sclerosis in an individual, includingassaying a test sample from the individual for the presence ofChlamydia, wherein the presence of Chlamydia in the sample indicates thepresence of multiple sclerosis.

[0006] In preferred embodiments, the Chlamydia is selected from thegroup consisting of Chlamydia pneumoniae, Chlamydia pecorum, Chlamydiapsittacci, and Chlamydia trachomatis, and the test sample is selectedfrom the group consisting of blood, serum, peripheral blood mononuclearcells, cerebrospinal fluid, urine, nasal secretion, and saliva.

[0007] In one embodiment, the test sample is assayed for the presence ofChlamydia by contacting cultured chlamydia-free indicator cells (e.g.,HL cells, H292 cells, HeLa cells, or Hep-2 cells) with the test sample;and then detecting the presence of Chlamydia in the cultured indicatorcells. The presence of Chlamydia in the cultured indicator cells isindicative of the presence of Chlamydia in the test sample.

[0008] The presence of Chlamydia in the cultured indicator cells can bedetected by detecting an antibody to Chlamydia (e.g, an antibody to aChlamydia elementary body antigen), a Chlamydia gene, or a Chlamydiaprotein in the test sample. The presence of the antibody, gene, orprotein is indicative of the presence of Chlamydia in the test sample.In one embodiment, the test sample is incubated under disulfide reducingconditions (e.g., incubating a disulfide reducing agent such as2,3-dimercaptosuccinic acid, penicillamine, β-lactams, dithiotreitol,mercaptoethylamine, or N-acetylcysteine) prior to detecting the presenceof Chlamydia.

[0009] In another aspect, the invention features a method of isolatingelementary bodies from a receptacle containing elementary bodies. Themethod includes treating the receptable with trypsin/EDTA to releaseelementary bodies adhered to the receptacle; and then concentrating theelementary bodies by centrifugation or filtration.

[0010] In still another aspect, the invention features a method ofreleasing DNA from elementary bodies, the method including incubatingthe elementary bodies under disulfide reducing conditions and digestingthe elementary bodies with a protease.

[0011] In yet another aspect, the invention features a method oftreating an individual diagnosed to have multiple sclerosis, includingadministering to the individual an effective amount of at least oneanti-chlamydial agent. In one embodiment, the individual is administeredthe anti-chlamydial agent until the individual tests negative forelementary body phase Chlamydia, replicating phase Chlamydia, andcryptic phase Chlamydia. In another aspect, the individual isadministered the anti-chlamydial agent for at least 45 days. Theadminstration can be continued for longer periods, and it may bepreferable to continue the treatment for at least 90 days, at least 180days, or even for one year or more.

[0012] Preferable anti-chlamydial agents include rifamycins, azalides,macrolides, ketolides, streptogramins, ampicillin, amoxicillin,nitroimidazoles, nitrofurans, quilolones, fluoroquinolones,sulfonamides, isonicotinic congeners, and tetracyclines.

[0013] In one embodiment, the individual is also administered aneffective amount of an agent that increases inducible nitric oxidesynthase (iNOS) activity, such as a type-1 interferon (e.g.,α-interferon or β-interferon), a synthetic type-1 interferon analog, ora hybrid type-1 interferon. Preferably, the type-1 interferon analog orhybrid binds to the same receptor as a naturally-occurring type-1interferon. In another embodiment, the individual is administered atleast two anti-chlamydial agents.

[0014] In yet another aspect, the invention features a method oftreating an individual diagnosed to have multiple sclerosis, includingadministering to the individual (i) a rifamycin; and (ii) a compoundselected from the group consisting of azalides, macrolides, ketolides,and streptogramins. In addition, the individual can optionally beadministered ampicillin, amoxicillin, probenecid, a nitroimidazole, anitrofuran, or any combination thereof.

[0015] In another aspect, the invention features a method of treating anindividual diagnosed to have multiple sclerosis, including administeringto the individual one of the following combinations: a rifamycin,ampicillin or amoxicillin, and probenecid; a quinolone or afluoroquinolone and a rifamycin; a rifamycin, a sulfonamide, and anisonitotinic congener; or a rifamycin and a tetracycline. The individualcan also be administered an effective amount of a compound thatincreases iNOS activity (e.g., β-interferon).

[0016] The administration is preferably continued until the individualtests negative for elementary body phase Chlamydia, replicating phaseChlamydia, and cryptic phase Chlamydia, or for at least 45 days.

[0017] In still another aspect, the invention features a pharmaceuticalcomposition that includes one of the following combinations: arifamycin, ampicillin or amoxicillin, and probenecid; a quinolone or afluoroquinolone and a rifamycin; a rifamycin, a sulfonamide, and anisonitotinic congener; or a rifamycin and a tetracycline. Thecomposition can optionally include a compound that increases iNOSactivity (e.g., β-interferon).

[0018] In yet another aspect, the invention features a kit that includesan anti-chlamydial agent and a compound that increases iNOS activity. Ina preferred embodiment, the compound that increases iNOS activity is atype-1 interferon (e.g., β-interferon), a synthetic type-1 interferonanalog, or a hybrid type-1 interferon, wherein the type-1 interferonanalog or hybrid binds to the same receptor as a naturally-occurringtype-1 interferon. In another preferred embodiment, the anti-chlamydialagent is selected from the group consisting of rifamycins, azalides,macrolides, ketolides, streptogramins, ampicillin, amoxicillin,nitroimidazoles, quilolones, fluoroquinolones, sulfonamides,isonicotinic congeners, and tetracyclines.

[0019] In still another aspect, the invention features a method fordetermining whether a candidate compound is a potential drug for thetreatment of a disease caused or exacerbated by chlamydial infection,the method including the steps of: (a) infecting a non-human animal(e.g., a non-human mammal) with Chlamydia; (b) administering a candidatecompound to the animal; and (c) assaying for the presence of achlamydial infection in a test sample from the mammal. A decrease in thelevel of infection, relative to the level of infection of a controlanimal infected with chlamydia but not administered a candidatecompound, identifies the candidate compound as a potential drug for thetreatment of disease caused or exacerbated by a chlamydial infection.Preferably, the animal is a non-human mammal and brain of the mammal isinfected with Chlamydia.

[0020] In preferred embodiments, the Chlamydia is selected from thegroup consisting of Chlamydia pneumoniae, Chlamydia pecorum, Chlamydiapsittacci, and Chlamydia trachomatis, and the test sample is selectedfrom the group consisting of blood, serum, cerebrospinal fluid, urine,nasal secretion, and saliva. In another preferred embodiment, thedisease is multiple sclerosis. The animal can be, for example, a mouse,rat, rabbit, or amoeba.

[0021] In one embodiment, the test sample is assayed for the presence ofChlamydia by contacting cultured chlamydia-free indicator cells (e.g.,HL cells, H292 cells, HeLa cells, or Hep-2 cells) with the test sample;and then detecting the presence of Chlamydia in the cultured indicatorcells. The presence of Chlamydia in the cultured indicator cells isindicative of the presence of Chlamydia in the test sample.

[0022] The presence of Chlamydia in the cultured indicator cells canalso be detected by detecting an antibody to Chlamydia (e.g, an antibodyto a Chlamydia elementary body antigen), a Chlamydia gene, or aChlamydia protein in the test sample. The presence of the antibody,gene, or protein is indicative of the presence of Chlamydia in the testsample. In one embodiment, the test sample is incubated under disulfidereducing conditions (e.g., incubating a disulfide reducing agent such as2,3-dimercaptosuccinic acid, penicillamine, β-lactams, dithiotreitol,mercaptoethylamine, or N-acetylcysteine) prior to detecting the presenceof Chlamydia.

[0023] In a related aspect, the invention features a second method fordetermining whether a candidate compound is a potential drug for thetreatment of multiple sclerosis. This method includes the steps of: (a)infecting the brain of a non-human mammal (e.g., a rat, mouse, orrabbit) with Chlamydia; (b) administering a candidate compound to themammal; and (c) assaying for the loss of white matter in the brain ofthe mammal, wherein a decrease in the loss of white matter, relative tothe loss of white matter in a control mammal infected with chlamydia butnot administered any candidate compound, identifies the candidatecompound as a potential drug for the treatment of multiple sclerosis.

[0024] By “Chlamydia” or “chlamydial cell” is meant any organism of theorder Chlamydiales. Examples include, but are not limited to, C.psittacci, C. trachomatis, C. pecorum, C. abortus, C. caviae, C. felis,C. suis, C. muridarum, WSU-86-1044, Parachlamydia acanthamoebae, andSimkania negevensis. By “chlamydial infection” is meant an infection ofa cell by a chlamydial cell.

[0025] By “indicator cell” is meant a cell capable of being infected bya Chlamydia cell. Preferred indicator cells include HL cells, H292cells, HeLa cells, and Hep-2 cells, which have been shown to be free ofchlamydial infection.

[0026] By “long-term therapy” is meant the treatment of a disease (e.g.,MS) for at least 45 days, more preferably for at least 60 days or even90 days, and most preferably for at least 120 days, 180 days, or for ayear or more. The long-term therapy can be continued for a given length,or can be stopped when a patient tests negative for elementary bodyphase Chlamydia, replicating phase Chlamydia, and cryptic phaseChlamydia (e.g., by PCR of a disulfide reducing agent-treated samplefrom the patient).

[0027] It may be desirable to change one or all of the drugs in themiddle of the long-term therapy. Changes in drug combinations may be formany reasons, such as to reduce side effects or cost to the patient, orin response to a change in the patient's condition or degree ofinfection. Moreover, while it is preferable that the therapy iscontinuous, it is understood that interruption for as much as two weeksor even a month may be desirable or necessary. For example, anindividual may take drug combination A for 30 days, stop therapy for twoweeks, and then resume therapy (switching to drug combination B) for anadditional 30 days. Interrupted therapy and therapy in which one or moredrugs are added or removed are each considered to be long-term therapyif the number of days of therapy (i.e., excluding the days in which nodrugs for the treatment of MS were administered) is at least 45.

[0028] By “anti-chlamydial agent” is meant an agent that results in adecrease in the viability or replication of chlamydial cells at aconcentration that would not be substantially detrimental to the cellsin which the chlamydial cells were contained. Preferably, theanti-chlamydial agent decreases the viability or replication ofchlamydial cells by at least 50%, more preferably by at least 75% andmost preferably by at least 90% or even 95%. Preferred anti-chlamydialagents include, without limitation, rifamycins, azalides, macrolides,ketolides, streptogramins, ampicillin, amoxicillin, nitroimidazoles,quilolones, fluoroquinolones, sulfonamides, isonicotinic congeners, andtetracyclines.

[0029] The present invention provides methods for the diagnosis of MSwith a significant reduction in cost. In addition, these diagnosticassays provide objective data concerning the course of the disease and,thus, the ability to monitor disease progress and the effectiveness oftherapy. The invention also provides methods and reagents for thetreatment of a patient diagnosed with MS, as well as methods foridentifying new drugs for the such treatment.

[0030] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 is a schematic illustration showing visualization of a 446base pair region of the 16S rRNA gene of Chlamydia pneumoniae (alsoreferred to as Chlamydophila pneumoniae) amplified by a nested PCRprocedure and followed by Southern hybridization with adigoxigenin-labeled specific probe. The gels represent cerebrospinalfluid (CSF) from 17 patients with relapsing remitting MS and 13 patientswith other neurological diseases (OND) controls. The gels includequality control markers. Lane P represents a positive control of C.pneumoniae (VR1310, American Type Culture Collection (ATCC); Manassas,Va.) while lane C represents a distilled water negative control that hasbeen subjected to the entire PCR procedure.

[0032]FIG. 2 is a schematic illustration showing ELISA results ofanti-IgG and anti-IgM antibodies in CSF to elementary body (EB) antigensof C. pneumoniae in MS patients and controls. Antibody index isrepresented as the ratio of OD units measured by ELISA in patient groupover OD units of CSF from five pooled normal CSF samples to EB antigensof C. pneumoniae. In all experiments, 1 μg of immunoglobulin was addedto microtiter wells.

[0033] FIGS. 3A-3D are a series of schematic illustrations showingaffinity-driven immunoblot studies on four MS patients. In each figure,lanes 1-4 represent the banding pattern of oligoclonal antibodiesfollowing affinity-driven transfer onto untreated (lane 1), C.pneumoniae antigen-coated (lane 2), measles-antigen coated (lane 3), orHSV-1 antigen-coated (lane 4) nitrocellulose membranes and probed withanti-human Ig antibody.

[0034] FIGS. 4A-4D are a series of schematic illustrations showingaffinity-driven immunoblot studies on four OND patients. FIGS. 4A and 4Brepresent SSPE patients #1 and #2, respectively; FIG. 4C represents apatient with CNS syphilis; and FIG. 4D represents a patient with CNSvasculitis. In each figure, lanes 1-3 represent the banding pattern ofoligoclonal antibodies following affinity-driven transfer onto untreated(lane 1), measles-antigen coated (lane 2), or C. pneumoniaeantigen-coated (lane 3) membranes and detection with anti-human IgGantibody.

[0035] FIGS. 5A-5J are a series of schematic illustrations showingadsorption studies on CSF immunoglobulins to EB antigens of C.pneumoniae, measles, HSV-1, and MBP for 10 patients with progressive MS.For each individual patient, the left two lanes represent IEF gelpatterns for 0.8 μg Ig of unmanipulated serum and CSF, respectively,while the right lanes represent the IEF gel patterns followingincubation with antigens as labeled.

[0036] FIGS. 6A-6E are a series of schematic illustrations showingadsorption studies on CSF immunoglobulins to EB antigens of C.pneumoniae, measles, HSV-1, and MBP for five patients with relapsingremitting MS. For each individual patient, the left two lanes representIEF gel patterns for 0.8 μg Ig of unmanipulated serum and CSF, while theright lanes represent the IEF gel patterns following incubation withantigens. In three patients, the adsorption following incubation with C.pneumoniae is incomplete (FIGS. 6A-6C; Arrows indicate some bands of thecathodal antibodies that are adsorbed by C. pneumoniae antigens). In twopatients, no adsorption of CSF immunoglobulin by C. pneumoniae antigenis seen (FIGS. 6D and 6E).

[0037] FIGS. 7A-7F are a series of schematic illustrations showing IEFgel patterns following adsorption studies on CSF immunoglobulins forSSPE (FIGS. 7A-7C), CNS syphilis (FIG. 7D), CNS vasculitis (FIG. 7E),and chronic meningitis (FIG. 7F).

[0038]FIGS. 8A and 8B are schematic illustrations showing dose kineticsfor induction of iNOS (expressed as NO levels in supernatants) in murinemacrophage cultures following exposure to either EB antigens (FIG. 8A)or purified recombinant major outer membrane protein (MOMP) (FIG. 8B).

[0039]FIG. 9 is a schematic illustration showing enhancement of NOlevels in macrophage cultures exposed to EB antigen (2 μg/ml) followingpre-incubation with murine β-IFN.

[0040]FIG. 10 is a schematic illustration showing that increases in NOlevels are mediated by β-IFN. β-IFN was inactivated with specific sheepanti-mouse β-IFN antibody in amounts sufficient to neutralize 10 U ofβ-IFN. The amount of control sheep immunoglobulin added equaled Igconcentrations present in anti-sheep antibody that had the capacity toneutralize 100 U of β-IFN.

[0041]FIGS. 11A and 11B are schematic illustrations showing thatenhancement of NO levels in macrophage cultures exposed to purifiedrMOMP (FIG. 11A) or LPS (FIG. 11B) is also mediated by β-IFN.

[0042]FIGS. 12A and 12B are schematic illustrations showing the resultsof an RT-PCR assay for the presence of iNOS2 gene products in murinemacrophage cultures after exposure to EB antigens and purified rMOMP.

[0043]FIGS. 13A and 13B are schematic illustrations showing dosekinetics for induction of IL-12/p40 production after exposure to EBantigens (FIG. 13A) or purified rMOMP (FIG. 13B).

[0044]FIG. 14 is a schematic illustration showing inhibition ofproduction of IL-12/p40 in macrophage cultures pretreated with β-IFN andaddition of EB antigens.

[0045]FIGS. 15A and 15B are schematic illustrations showing anti-β-IFNantibody reverses the inhibition of β-IFN on IL-12/p40 productionfollowing addition of EB antigens (FIG. 15A) or rMOMP (FIG. 15B).

DETAILED DESCRIPTION OF THE INVENTION

[0046]C. pneumoniae belongs to the order Chlamydiales (the members ofwhich are herein referred to collectively as Chlamydia). Members of thisorder are obligately intracellular pathogens that are infectious tohumans and other vertebrates. Other species currently recognized includeC. psittacci, C. trachomatis, and C. pecorum. C. psittacci is known toinfect microglial cells, while C. pecorum in cattle causes a syndromeknown as sporadic bovine encephalomyelitis, for which detailedneuropathologic data are lacking (Storz J., Chlamydia and ChlamydialInduced Diseases. Springfield, Ill.: Charles C. Springer, 1971: 358). C.trachomatis and C. pneumoniae are pathogenic primarily to humans and arerecognized to cause latent disease. Meningoencephalitis and otherneurological complications have been described in patients withinfections due to C. psittaci and C. trachomatis (Korman et al., Clin.Infect. Dis. 25:847-851, 1997). In addition, the Chlamydiales orderincludes C. abortus, C. caviae, C. felis, C. suis, C. muridarum,WSU-86-1044, Parachlamydia acanthamoebae, and Simkania negevensis(Everett et al. Intl. J. System. Bacteriol. 49:415-440, 1999).

[0047] Diagnostic Assays

[0048] We have demonstrated a strong correlation between the presence ofC. pneumoniae in the CSF of patients with MS by cell culture, polymerasechain reaction (PCR), and immunological methods. C. pneumoniae wasisolated from CSF cultures and also was identified in CSF by PCRamplification of the ompA gene of C. pneumoniae. Moreover, CSF titers ofIgM and IgG against C. pneumoniae EB antigens were elevated as measuredby ELISA methodologies. The specificity of this antibody response for C.pneumoniae was shown by Western blot assays. PCR data in which CSFsamples from MS patients and other neurologic diseases (OND) controlswere analyzed for the 16S rRNA gene of C. pneumoniae using a nested PCRprocedure followed by Southern hybridization with a digoxigenin labeledspecific probe also established a link between MS and C. pneumoniaeinfection. Moreover, IEF/affinity-driven immunoblot assays show that thecationic antibodies in MS patients react to C. pneumoniae EB antigens.

[0049] As the presence of Chlamydia correlates with the presence of MS,the invention features a method for diagnosing a patient with MS. In themethods of the invention, a test sample from an individual, such as anindividual who is suspected of having MS, is used. The test sample caninclude blood, serum, cerebrospinal fluid, urine, nasal secretion,saliva, or any other bodily fluid or tissue, or antibodies or nucleicacids isolated from one of the foregoing samples.

[0050] The test sample can be assayed for the presence or absence ofChlamydia by culturing the test sample with indicator cells. Theindicator cells can be any cells which are capable of being infected byChlamydia, and which preferably have been shown to be free of infectionby Chlamydia and free of elementary bodies of Chlamydia. Representativeindicator cells include HL cells, H292 cells, HeLa cells, Hep-2 cells,or any other cell line capable of supporting replication of Chlamydia.The indicator cells are cultured in the presence of the test sample andthen assayed for the presence or absence of Chlamydia by an appropriatemethod, such as by exposing the cultured indicator cells to a detectableantibody that is specific for Chlamydia. The presence of Chlamydia inthe cultured indicator cells indicates the presence of Chlamydia in thetest sample.

[0051] The test sample can also be assayed for the presence or absenceof Chlamydia by detecting the presence or absence of a Chlamydia gene(e.g., a gene encoding MOMP, OMP-B, GRO-ES, GRO-EL, DNAK, 16S RNA, 23SRNA, ribonuclease-P, the 76 kD attachment protein, or a KDO-transferase)in the test sample. For example, the test sample can be assayed for thepresence or absence of the Chlamydia gene by Southern hybridizationusing a detectable probe for the appropriate gene. Alternatively, thetest sample can be assayed using quantitative PCR or RT-PCR (e.g., byusing a LightCycler™ (Idaho Technology Inc., Idaho Falls, Id.) andfluorescent LightCycler™ probes). The presence of the Chlamydia gene inthe test sample is indicative of the presence of Chlamydia in the testsample. To facilitate assaying a test sample for the presence or absenceof Chlamydia by detecting the presence or absence of a Chlamydia gene,the test sample can be subjected to methods to enhance isolation ofChlamydia elementary bodies from the test sample and to release DNA fromthe elementary bodies. For example, elementary bodies have a tendency toadhere to the walls of a receptacle containing them; the elementarybodies can be removed from the receptacle by treating the receptaclecontaining the elementary bodies with trypsin/EDTA, thereby releasingelementary bodies that adhered to the receptacle; and then concentratingthe released elementary bodies, such as by centrifugation or filtration.To release DNA from elementary bodies, the elementary bodies areincubated under disulfide reducing conditions, such as incubating theelementary bodies with a disulfide reducing agent such as dithiothreitol(DTT) or 2-mercaptoethanol; and digesting the elementary bodies with aprotease.

[0052] The test sample can also be assayed for the presence of Chlamydiaby detecting the presence of a protein from Chlamydia. For example, thepresence of a MOMP protein in the test sample can be detected throughthe use of ELISA methodologies with an antibody that specificallyrecognizes the MOMP protein. Alternatively, the test sample may beassayed for the presence of Chlamydia by detecting the presence ofantibodies to Chlamydia, or to Chlamydia EB antigens, in the testsample. The presence of Chlamydia protein or antibodies to Chlamydia orChlamydia EB antigens in the test sample is indicative of the presenceof Chlamydia in the test sample. In either of these methods, ChlamydiaEB antigens can be prepared by incubating Chlamydia EBs under disulfidereducing conditions, such as in the presence of at least one disulfidereducing agent such as DTT or 2-mercaptoethanol, or another disulfidereducing agent. The presence of proteins or antibodies may be detectedby appropriate methods such as by ELISA, Western blot, or isoelectricfocusing.

[0053] The diagnostic methods described herein are useful for detectingor confirming the disease in a patient, as well as for monitoring theprogress of the disease. Disease monitoring is useful, for example, fordetermining the efficacy of a particular therapy.

[0054] Diagnostic Reagents

[0055] The invention also provides a diagnostic reagent kit includingone or more containers filled with one or more of the ingredients usedin the assays of the invention. Optionally associated with such a kitcan be a notice in the form prescribed by a governmental agencyregulating the manufacture, use, or sale of diagnostic products, whichreflects approval by the agency of manufacture, use or sale for humanadministration. The kit can be labeled with information regarding modeof administration, sequence of execution (e.g., separately,sequentially, or concurrently), or the like. The kit can be a singleunit assay or it can be a plurality of unit assays. In particular, theagents can be separated, mixed together in any combination, present in asingle vial or tablet. For the purpose of this invention, a unit assayis intended to mean material sufficient to perform only a single assay.Therapy In addition to demonstrating that C. pneumoniae infectioncorrelates with MS, we have also found that patients with MS that weretreated with anti-chlamydial agents showed improved Expanded DisabilityStatus Scale (EDSS; Kurtzke, Neurology 33:1444-1152, 1983) scores. Thus,it is highly likely that chlamydial infection causes or exacerbates MS.We have also identified combination therapy regimens that, because ofthe phase of Chlamydia targeted by each drug, are particularly suitedfor the treatment of MS.

[0056] A) Anti-Chlamydial Agents

[0057] Chlamydia are obligate intracellular bacterial parasites ofeukaryotic cells. Members of this order have a unique biphasicdevelopment cycle with distinct morphological and functional forms. Thisdevelopmental growth cycle alternates between (i) intracellular lifeforms of which two are currently recognized: an intracellular form whichcan exist as a metabolically-active, replicating organism known as thereticulate body (RB) or a persistent, nonreplicating form known as thecryptic body; and (ii) an extracellular EB form that is infectious andmetabolically-inactive.

[0058] EBs are small (300 to 400 nm) infectious spore-like forms whichare resistant to a variety of physical insults such as enzymedegradation, sonication, and osmotic pressure. This physical stabilityis likely a result of extensive disulfide cross-linking of thecysteine-rich MOMP. Under the oxidizing conditions of the extracellularmilieu of the host, the outer membrane of EBs is relatively impermeableand indestructible.

[0059] A number of effective agents that are specifically directedagainst the initial phase of chlamydial infection (i.e., the transitionof the chlamydial EB to a reticulate body (RB)) have been identified.These include compounds in the rifamycin class and act againstDNA-dependent RNA polymerase, which is present when the EB begins totransform into the RB phase. Inhibition of this chlamydial DNA-dependentRNA polymerase prevents this transition.

[0060] A number of effective agents that are specifically directedagainst the cryptic growth phase have also been identified. This crypticgrowth phase, unlike that of the replicating chlamydial microorganism,which uses host cell energy, involves electrons and electron transferproteins, as well as nitroreductases. Accordingly, the initial phase ofChlamydia infection is susceptible to the antimicrobial effects ofnitroimidazoles, nitrofurans, and other agents directed againstanaerobic metabolism in bacteria. Nitroimidazoles and nitrofurans aresynthetic antimicrobial agents that are grouped together because bothare nitro (NO₂—) containing ringed structures and have similarantimicrobial effects. These effects require degradation of the agentwithin the microbial cell such that electrophilic radicals are formed.These reactive electrophilic intermediates then damage nucleophilicprotein sites including ribosomes, DNA, and RNA. Nitroimidazoles andnitrofurans were not previously considered to possess antimicrobialactivity against Chlamydia. This apparent lack of antimicrobialactivity, however, is due to the fact that conventional susceptibilitytesting methods only test for effect on the replicating form ofChlamydia, and do not measure the presence of other forms of Chlamydia.

[0061] Examples of suitable nitroimidazoles include, but are not limitedto, metronidazole, tinidazole, bamnidazole, benznidazole, flunidazole,ipronidazole, misonidazole, moxnidazole, ronidazole, sulnidazole, andtheir metabolites, analogs, and derivatives thereof. Metronidazole ismost preferred. Examples of nitrofurans that can be used include, butare not limited to, nitrofurantoin, nitrofurazone, nifurtimox,nifuratel, nifuradene, nifurdazil, nifurpirinol, nifuratrone,furazolidone, and their metabolites, analogs, and derivatives thereof.Nitrofurantoin is preferred within the class of nitrofurans. Throughoutthis application and for purposes of this invention, “metabolites” areintended to embrace products of cellular metabolism of a drug in thehost (e.g., human or animal) including, but not limited to, theactivated forms of prodrugs. The terms “analogs” and “derivatives” areintended to embrace isomers, optically active compounds, and anychemical or physical modification of an agent, such that themodification results in an agent having similar or increased, but notsignificantly decreased, effectiveness against Chlamydia, compared tothe effectiveness of the parent agent from which the analog orderivative is obtained. This comparison can be ascertained usingsusceptability testing. Cells to be treated can already be crypticallyinfected or they can be subjected to stringent metabolic orenvironmental conditions which cause or induce the replicating phase toenter the cryptic phase. Such stringent conditions can include changingenvironmental/culturing conditions in the instance where the infectedcells are exposed to γ-interferon; or by exposing cells to conventionalantimicrobial agents (such as macrolides and tetracyclines) which inducethis cryptic phase of chlamydial infection in human host cells.

[0062] A class of anti-chlamydial agents that is effective against thereplicating and cryptic stationary phases of Chlamydia (and possiblyagainst some other stages of the cryptic phase) have been identified.This class of agents includes ethambutol and isonicotinic acidcongeners, which include isoniazid (INH), isonicotinic acid (also knownas niacin), nicotinic acid, pyrazinamide, ethionamide, and aconiazide.INH is the most preferred compound in this class. Although thesecompounds were previously considered effective only for mycobacterialinfections, we have discovered that these, agents, in combination withother antibiotics, are effective against Chlamydia. It is believed thatthe isonicotinic acid congeners target the constitutive production ofcatalase and peroxidase, which is a characteristic of microorganisms,such as mycobacteria, that infect monocytes and macrophages. Chlamydiacan also successfully infect monocytes and macrophages.

[0063] Using INH to eradicate Chlamydia from macrophages and monocytessubsequently assists these cells in their role of fighting infection.These agents appear to be less effective in vitro against the crypticphase. Thus, ethambutol, INH, and other isonicotinic acid congenersideally should be used in combination with agents that target otherphases of the chlamydial life cycle. These isonicotinic acid congenersare nevertheless excellent agents for the long term therapy ofchronic/systemic chlamydial infection.

[0064] Adverse conditions, such as limited nutrients, antimicrobialagents, and the host immune response, produce a stringent response inChlamydia. This stringent response alters the morphological state of theintracellular microorganism and creates dormant forms, including theintracellular EB, which then can cryptically persist until itsdevelopmental cycle is reactivated. Conversely, the host cell may lyseand allow the EBs to reach the extracellular milieu. Thus, it isnecessary to utilize a combination of agents directed toward the variouslife stages of Chlamydia and, in particular, against the elementary bodyfor successful management of infection.

[0065] During the chlamydial life cycle, it is known thatmetabolically-inactive spore-like EBs are released into theextracellular milieu. Although these released EBs are infectious, theymay not immediately infect nearby susceptible host cells untilappropriate conditions for EB infectivity are present. The result ofthis delay in infection is the extracellular accumulation ofmetabolically-inactive, yet infectious, EBs. This produces a second typeof chlamydial persistance referred to herein as EB “tissue/blood load.”This term is similar in concept to HIV load and is defined herein as thenumber of infectious EBs that reside in the extracellular milieu. Directmicroscopic visualization techniques, tissue cell cultures, andpolymerase chain reaction test methods have demonstrated that infectiousEBs are frequently found in the blood of apparently healthy animals,including humans. This phenomenon is clearly of great clinicalimportance in chlamydial infections as these metabolically-inactive EBsescape the action of current anti-chlamydial therapy which is directedonly against the replicating intracellular forms of Chlamydia. Thepresence of infectious extracellular EBs after the completion of shortterm, anti-replicating phase therapy for chlamydial infections has beenshown to result in intracellular infection relapse. Thus, the durationand nature of anti-chlamydial therapy required for management ofchlamydial infections is, in part, dictated by the extracellular load ofEBs. For purposes of this invention, short term therapy can beapproximately two to three weeks; long-term therapy, in contrast, maycontinue for one or several months (see below).

[0066] It is also believed that persistance of chlamydial infections maybe due in part to the presence of cryptic forms of Chlamydia within thecells. This cryptic intracellular chlamydial form apparently can beactivated by certain host factors such as cortisone (Yang et al.,Infect. and Immun., 39:655-658, 1983; Malinverni et al., J. Infect.Dis., 172:593-594, 1995). Anti-chlamydial therapy for chronic Chlamydiainfections must be continued until any intracellular EBs or otherintracellular cryptic forms have been activated and extracellular EBshave infected host cells. This reactivation/reinfection by chlamydialEBs clearly is undesirable as it prolongs the therapy of chlamydialinfections, as well as increases the opportunity for antimicrobialresistance to occur.

[0067] Physiochemical agents have been identified that can inactivatechlamydial EBs in their respective hosts by reducing disulfide bondswhich maintain the integrity of the outer membrane proteins of the EBs.For Chlamydia, disruption of the outer membrane proteins of EBs therebyinitiates the transition of the EB form to the RB form. When this occursin the acellular milieu where there is no available energy source, thenascent RB perishes or falls victim to the immune system. Thus,disulfide reducing agents that can interfere with this process aresuitable as compounds for eliminating EBs.

[0068] One such class of disulfide reducing agents are thiol-disulfideexchange agents. Examples of these include, but are not limited to,2,3-dimercaptosuccinic acid (DMSA; also referred to herein as“succimer”); D,L,-β,β-dimethylcysteine (also known as penicillamine);β-lactam agents (e.g., penicillins, penicillin G, ampicillin andamoxicillin, which produce penicillamine as a degradation product),cycloserine, DTT, mercaptoethylamine (e.g., mesna, cysteiamine,dimercaptol), N-acetylcysteine, tiopronin, and glutathione. Aparticularly effective extracellular anti-chlamydial agent within thisclass is DMSA, which is a chelating agent having four ionizablehydrogens and two highly charged carboxyl groups which prevent itsrelative passage through human cell membranes. DMSA thus remains in theextracellular fluid where it can readily encounter extracellular EBs.The two thiol (sulfhydryl) groups on the succimer molecule (DMSA) areable to reduce disulfide bonds in the MOMP of EBs located in theextracellular milieu. Penicillamine can also be used as a disulfidereducing agent to eliminate chlamydial EBs. The use of penicillamine,however, may cause undesirable side effects. Thus, as an alternative,those β-lactam agents which are metabolized or otherwise converted topenicillamine-like agents in vivo (i.e., these agents possess a reducinggroup) can be orally administered to the human or animal as a means ofproviding a controlled release of derivative penicillamine, bynon-enzymatic acid hydrolysis of the penicillin, under physiologicconditions. Clavulonic acid is not required for this hydrolysis or forusing β-lactam agents to create penicillamine in vivo.

[0069] As chlamydial RBs transform into EBs, they begin to utilizeactive transcription of chlamydial DNA and translation of the resultingmRNA. As such, these forms of Chlamydia are susceptible to currentlyused antimicrobial agents. The anti-chlamydial effectiveness of theseagents can be significantly improved by using them in combination withother agents directed at different stages of Chlamydia life cycle, asdiscussed herein.

[0070] Classes of suitable antimicrobial agents include, but are notlimited to, rifamycins (also known as ansamacrolides), quinolones,fluoroquinolones, chloramphenicol, sulfonamides/sulfides, azalides,cycloserine, macrolides, ketolides, and tetracyclines. Examples of theseagents which are members of these classes, as well as those which arepreferred, are illustrated below in Table 1. TABLE 1 Drug Class ExamplesPreferred Quinolones/Fluoroquinolones Ofloxacin LevofloxacinLevofloxacin Trovafloxacin Sparfloxacin Norfloxacin LomefloxacinCinoxacin Enoxacin Nalidixic Acid Fleroxacin Ciprofloxacin SulfonamidesSulfamethoxazole Sulfamethoxazole/ Trimethoprim Azalides AzithromycinAzithromycin Macrolides Erythromycin Clarithromycin ClarithromycinLincosamides Lincomycin Clindamycin Clindamycin TetracyclinesTetracycline Minocycline Doxycycline Minocycline MethacyclineOxytetracyline Rifamycins Rifampin Rifampin (Ansamacrolides) Rifabutin

[0071] Members of Chlamydia, including C. pneumoniae, were previouslyconsidered to be inhibited, and some killed, by the use of a singleagent selected from currently used antimicrobial agents such as thosedescribed above. We have found, however, that complete eradication ofChlamydia cannot be achieved by the use of any one of these agentsalone, unless the administration is of sufficient length (see below),because none are efficacious against all phases of the Chlamydia lifecycle and appear to induce a stringent response in Chlamydia, causingthe replicating phase to transform into cryptic forms and resulting in apersistent infection that can be demonstrated by PCR techniques whichassess the presence or absence of chlamydial DNA. Nevertheless, one ormore of these currently used agents, or another agent directed againstthe replicating phase of Chlamydia, should be included as one of thechlamydial agents in a combination therapy in order to slow or halt thetransition of the EB to the RB as well as to inhibit chlamydialreplication.

[0072] For the treatment of MS, the combinations of anti-chlamydialagents shown in Table 2 are preferred. TABLE 2 Combination Drug ClassPreferred 1 Rifamycin Rifampin Azalide Azithromycin Macrolide KetolideStreptogramin 2 Rifamycin Rifampin Ampicillin or Amoxicillin Probenecid3 Rifamycin Rifampin Azalide Macrolide Ketolide Ampicillin orAmoxicillin Azithromycin Probenecid 4 Rifamycin Rifampin AzalideAzithromycin Macrolide Ketolide Streptogramin Ampicillin or AmoxicillinProbenecid Nitroimidazole Metronidazole 5 Fluoroquinolone OfloxacinLevoflozacin Rifamycin Rifampin 6 Sulfonamide Sulfamethoxazole/Trimethoprim Rifamycin Rifampin Isonicotinic congener INH 7 RifamycinRifampin Tetracycline Minocycline

[0073] To any of the drug combinations, any or all of the followingcompounds can also be added: probenecid, disulfide reducing agents(e.g., penicillamine), statins (e.g., dantolene), type-1 interferons(e.g., α-IFN or β-IFN), and activators of iNOS activity.

[0074] B) Compounds that Increase iNOS Expression or Activity

[0075] Nitric oxide (NO) is a relatively unstable free radicalsynthesized from L-arginine by inducible nitric oxide synthase (iNOS)and is considered to play a role in containing and/or eradicatingintracellular pathogens. NO is implicated in a number of in vitro and invivo models of host resistance to intracellular pathogens such asLeishmania major, Toxoplasma gondii, Listeria monocytogenes, andMycobacterium tuberculosis. iNOS may also play a role in inhibitingreplication of C. trachomatis in epithelial cells. Moreover, disruptionof the iNOS gene in mice leads to dissemination of C.trachomatis-infected macrophages and delays the clearance of C.pneumoniae infections.

[0076] We have discovered that heat-killed EBs from C. pneumoniaeincrease iNOS expression, which, as described above, likely helpseradicate intracellular pathogens. Thus, any compound that increasesiNOS activity will likely reduce chlamydial infection and improve ormaintain neurological function in patients with MS. iNOS activity may bemeasured, for example, by measuring NO production, nitrate levels, orthe level of iNOS mRNA. Preferably, the increase in iNOS activity is byat least 10%, more preferably by at least 25%, and most preferably by50%, 100%, or more.

[0077] C) Type-1 Interferons

[0078] We have discovered that β-IFN increases iNOS activity. Based onthese findings, it is likely that any type-1 interferon would alsoincrease iNOS activity and, thus, be useful for the treatment of MS.

[0079] In accordance with the present invention, a type-1 interferon maybe a purified, naturally-occurring, or recombinant subtype, or it may bea hybrid of two or more subtypes or an analog thereof. Further, mixturescontaining any two or more of the above may be used in accordance withthe present invention. Many variations of the α-IFN and/or β-IFNsubtypes, hybrids, and/or analogs may be used. Furthermore, inaccordance with the present invention, the α-IFN and/or β-IFN mayoriginate from any mammalian species. Thus, for example, bovine β-IFNsubtypes may be used in human therapy.

[0080] First, α-IFN and/or β-IFN subtypes may be used which have alength of 166 amino acid units, and which have at least 60% of theconsensus sequence shown in Tables 1 and 2 of U.S. Pat. No. 5,780,021,respectively. The remaining portion of the consensus sequence and anyportion of or all of the non-consensus portions of any α-IFN or β-IFNmay be substituted by any other amino acid, whether naturally occurringor not. By the term “non-consensus” portion or “non-consensus” aminoacids is meant those amino acids which do not fall within the aminoacids which are sequentially common to α-IFN and/or β-IFNs as shown inTables 1 and 2 of U.S. Pat. No. 5,780,021. Thus, for example, any α-IFNsubtype from Table 1 and/or any β-IFN from Table 2 may be used as astarting model, and up to 40% of the consensus sequence may besubstituted and up to 100% of the non-consensus sequence may besubstituted by amino acids, such as, for example, glycine, alanine,valine, leucine, isoleucine, serine, threonine, cysteine, cystine,methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine,hydroxylysine, histidine, arginine, phenylalanine, tyrosine, andtryptophan, or even amithine or citrulline.

[0081] Second, α-IFN and/or β-IFN subtypes, hybrids, and/or analogs maybe used which are fewer than 166 amino acid residues. In accordance withthe present invention, the same rules will apply here as with the firstvariation above, except that the overall sequence length may beabbreviated to at least 70%, preferably at least 80% (132 or 133 units),and more preferably still to at least 90% (149 or 150 units).

[0082] Third, the α-IFN and/or β-IFN subtypes, hybrids, and/or analogsor mixtures thereof may be incorporated as an “active portion” into alarger polypeptide or protein of the formula:

ε-γ-ω

[0083] wherein γ is the “active portion” as defined above, and ε and ωeach independently represent from 0 to up to about 10,000 amino acids asdefined above, with the proviso that the polypeptide or protein has theactive portion, γ, topologically available at the surface of thepolypeptide or protein in the event that it is folded in athree-dimensional structure. The design of such structures, such that aparticular portion is available at the surface of the structure iswithin the skill of one in the art. Further, in reference to type-1interferons, the term “analog” means any active portion or sequencedescribed herein having at least 60% of the same amino acids in the samesequence as any sequence described in Table 1 or Table 2 of U.S. Pat.No. 5,780,021.

[0084] Generally, the term “interferon” refers to a family of proteinsthat confer non-specific resistance to a broad range of viralinfections, affect cell proliferation, and modulate immune responses.Three major interferons, α-, β- and γ- have been identified based uponantigenic and physico-chemical properties, the nature of the inducer,and the cellular source from which they are derived. α-IFN and β-IFN(known collectively as type-1 interferons), are structurally related andcompete for the same cell surface receptor. γ-IFN, known as type-2interferon, is structurally unrelated to type-1 IFNs and is acid labileand has a different cell surface receptor.

[0085] α-IFN refers to a family of highly homologous proteins thatinhibit viral replication and cellular proliferation and which modulateimmune responses. α-IFN is produced by many cells in the body, includingperipheral blood leukocytes or lymphoblastoid cells upon exposure tolive or inactivated virus, double-stranded RNA, or bacterial products.Moreover, there are multiple subtypes of α-IFN which contain 165-166amino acids and which have molecular weights of about 18,000 to 20,000daltons. β-IFN is a cytokine having antiviral, antiproliferative, andimmunomodulatory activities. Generally, β-IFN is a glycoproteincontaining 166 amino acids having a molecular weight of about 20,000daltons.

[0086] The amount of single subtype of α-IFN or β-IFN, hybrids, analogsor mixtures thereof administered per dose either prior to or after onsetof disease is about 1×10⁵ units to about 7.5×10⁷ units withadministrations being given from once per day to once per week. Amountsmay be used, however, which are less than 1×10⁵ units, such as 5×10⁴units or lower, or which are more than 7.5×10⁷ units, such as 1×10⁸units or higher. Of course, the precise amount used will vary, dependingupon the judgment of the attending physician, considering such factorsas the age, weight, and condition of the patient.

[0087] By “consensus sequence” is meant that sequence which is common toall α-IFN or β-IFN subtypes (see Tables 1 and 2 of U.S. Pat. No.5,780,021).

[0088] Table 1 of U.S. Pat. No. 5,780,021 provides a detailed sequencelisting of various α-IFN subtypes, showing a consensus sequence for all.In accordance with the present invention, any α-IFN subtype may be usedsingly or in admixture with others or as hybrids and/or analogs ormixtures thereof as long as it contains at least 60% of the consensussequence shown in Table 1 as described above or a sequence whichexhibits substantially the same α-IFN activity against autoimmunedisease as a sequence having at least that portion of the consensussequence.

[0089] Table 2 of U.S. Pat. No. 5,780,021 provides a comparison ofdetailed sequence listings for β-IFN of human, murine, and bovineorigin. In accordance with the present invention, any β-IFN subtype maybe used as long as it contains at least 60% of the consensus sequenceshown in Table 2 as described above or a sequence which exhibitssubstantially the same β-IFN activity against autoimmune disease as asequence having at least the consensus sequence.

[0090] Further, hybrid interferons may be constructed and used. Suchhybrid interferons are well known (see, for example, Pestka et al., J.Biol. Chem. 257:11497-11502, 1982).

[0091] Modes of Administration

[0092] The agents of the present invention can be formulated in aphysiologically acceptable vehicle in a form which will be dependentupon the method by which it is administered. In one aspect, theinvention pertains to a combination of agents, each of which is targetedagainst a different phase of the chlamydial life cycle or enhances theanti-chlamydial activity of other agents. The combination of agents canbe used in the management of chlamydial infection or prophylaxis thereofto prevent recurrent infection. The combination of agents can be in theform of an admixture, as a kit, or individually, and/or by virtue of theinstruction to produce such a combination. It is understood thatcombination therapy can include multiple agents that are effectivewithin a particular phase of the chlamydial life cycle. The combinationof agents can also include immunosuppressants, anti-inflammatory agents,vitamin C, or combinations thereof.

[0093] The therapeutic methods described herein can be used toameliorate or stabilize conditions/symptoms associated with MS, when thedisease is caused or aggravated by chlamydial infection. Compounds andagents described herein can be administered to an individual usingstandard methods and modes which are typically routine for the diseasestate. While any mammal may be treated, such as dogs, cats, cows, pigs,horses, or poultry, it is particularly desirable that the mammal treatedbe human.

[0094] Combinations of agents of this invention can be used for themanufacture of a medicament for simultaneous, separate, or sequentialuse in managing chlamydial infection or prophylaxis thereof. The agentscan also be used for the manufacture of a medicament for the treatmentof MS. The agents can be administered subcutaneously, intravenously,parenterally, intraperitoneally, intradermally, intramuscularly,topically, enteral (e.g., orally), sublingually, rectally, nasally,buccally, vaginally, by inhalation spray, by drug pump or via animplanted reservoir in dosage formulations containing conventionalnon-toxic, physiologically acceptable carriers or vehicles. Thepreferred method of administration is by oral delivery. The form inwhich it is administered (e.g., syrup, elixir, capsule, tablet,solution, foams, emulsion, gel, sol) will depend in part on the route bywhich it is administered. For example, for mucosal (e.g., oral mucosa,rectal, intestinal mucosa, bronchial mucosa) administration, nose drops,aerosols, inhalants, nebulizers, eye drops, or suppositories can beused. The compounds and agents of this invention can be administeredtogether with other biologically active agents.

[0095] In a specific embodiment, it may be desirable to administer theagents of the invention to the brain; this may be achieved by, forexample, and not by way of limitation, local infusion during surgery, byinjection, by means of a catheter, by means of a suppository, or bymeans of an implant (e.g., a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes or fibers). When it isdesirable to direct the drug to the central nervous system, techniqueswhich can opportunistically open the blood brain barrier for a timeadequate to deliver the drug there through can be used. For example, acomposition of 5% mannitose and water can be used.

[0096] The present invention also provides pharmaceutical compositions.Such compositions include a therapeutically (or prophylactically)effective amount of the agent, and a pharmaceutically acceptable carrieror excipient. Such a carrier includes but is not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The carrier and composition can be sterile. The formulationshould suit the mode of administration.

[0097] Suitable pharmaceutically acceptable carriers include, but arenot limited to, water, salt solutions (e.g., NaCl), alcohols, gumarabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters,hydroxymethylcellulose, and polyvinyl pyrolidone. The pharmaceuticalpreparations can be sterilized and if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. The composition can be a liquidsolution, suspension, emulsion, tablet, pill, capsule, sustained releaseformulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,polyvinyl pyrollidone, sodium saccharine, cellulose, magnesiumcarbonate, etc.

[0098] The composition can be formulated in accordance with the routineprocedures as a pharmaceutical composition adapted for intravenousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water, saline ordextrose/water. Where the composition is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients may be mixed prior to administration.

[0099] Agents described herein can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0100] The amount of agents which will be effective in the treatment ofa particular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

[0101] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions and/or adjunct therapiesof the invention. Optionally associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use of salefor human administration. The pack or kit can be labeled withinformation regarding mode of administration, sequence of drugadministration (e.g., separately, sequentially or concurrently), or thelike. The pack or kit may also include means for reminding the patientto take the therapy. The pack or kit can be a single unit dosage of thecombination therapy or it can be a plurality of unit dosages. Inparticular, the agents can be separated, mixed together in anycombination, present in a single vial or tablet. Agents assembled in ablister pack or other dispensing means is preferred. For the purpose ofthis invention, unit dosage is intended to mean a dosage that isdependent on the individual pharmacodynamics of each agent andadministered in FDA approved dosages in standard time courses.

[0102] The invention will be further illustrated by the followingnon-limiting examples of diagnostic and therapeutic methods.

EXAMPLE 1 Identification of the Presence of C. pneumoniae in Individualswith MS

[0103] A) Methods

[0104] Patient Population

[0105] The study evaluated 17 patients with relapsing remitting MS(4M/13F, mean age 31 years) at the time of diagnosis of clinicallydefinite disease (Table 3) and 20 patients with progressive MS (10M/10F,mean age 40 years) (Table 4). Among the 17 relapsing remitting MSpatients, two patients were on β-IFN that was instituted four weeks and16 weeks, respectively, prior to the enlistment of these patients forthe lumbar puncture. Both patients (#5 and #14) were also recoveringfrom a recent clinical worsening. The remaining 15 patients were not onany immunosuppressive or immunomodulatory drugs. All except three ofthese 17 MS patients had oligoclonal bands in the CSF (Table 3). Amongthe patients with progressive disease, two had primary progressivedisease, four had relapses with sequelae (relapsing progressivedisease), and the remaining 14 had secondary progressive disease. Fourof these 20 MS patients were on β-IFN, and three were on methotrexate atthe time the lumbar puncture was performed. All except two of these 20MS patients had oligoclonal bands in the CSF (Table 4). Twenty-sevenpatients (12M/15F, mean age 39 years) with other neurological diseases(OND) were selected as controls (Table 5). Of these, 19 had CSFabnormalities (i.e., increased CSF protein and/or increase in CSFlymphocytes) consistent with either a break in the blood-CSF orblood-brain barrier. One patient (#6) with chronic meningitis of unknownetiology had oligoclonal bands in the CSF. Of the remaining seven ONDcontrol patients with normal CSF profiles, two were diagnosed withcerebrovascular disease and one case each was seen with brain abscess,Hashimoto's encephalopathy, polyneuropathy, Wernike-Korsakoff'sencephalopathy, and a syndrome consistent with vasculitis and stroke.TABLE 3 Time from Onset of 1^(st) CSF Protein Patient Age/ symptom toConcentration; CSF CSF Ig Oligoclonal CSF CSF Number Sex Dx EDSS Cellcount Index Bands Culture PCR/S 1 26/F 1 year 1.5 32 mg/dl;  4 cell/μl1.28 Present Negative Positive 2 47/F 2 months 3.0 47 mg/dl;  7 cell/μl1.09 Present Positive Positive 3 22/F 2 years 1.5 40 mg/dl; 11 cell/μl0.60 Present Negative Positive 4 51/F 3 years 1.5 32 mg/dl;  0 cells/μl0.61 Present Negative Positive 5 22/F 6 months 6.0 34 mg/dl; 10 cell/μl0.90 Present Positive Positive 6 20/F 3 months 1.0 24 mg/dl;  9 cell/μl1.42 Present Negative Positive 7 49/M 1 year 3.5 57 mg/dl;  4 cell/μl1.44 Present Positive Positive 8 50/M 6 months 3.5 86 mg/dl;  2 cell/μl1.22 Present Positive Positive 9 39/F 6 months 2.0 53 mg/dl;  0 cells/μl0.55 Absent Negative Positive 10 29/F 12 years 3.5 22 mg/dl;  0 cells/μl0.48 Absent Negative Positive 11 28/F 4 months 1.5 68 mg/dl;  1 cell/μl0.5 Absent Positive Positive 12 27/F 4 years 2.5 45 mg/dl;  2 cells/μl1.97 Present Negative Positive 13 49/F 1.5 years 2.5 55 mg/dl;  2cell/μl 1.60 Present Negative Positive 14 47/M 2 years 5.5 82 mg/dl; 14cells/μl 0.67 Present Negative Positive 15 26/F 6 months 3.0 20 mg/dl; 6 cell/μl 1.19 Present Positive Positive 16 40/F 22 years 2.0 33 mg/dl; 1 cell/μl 0.88 Present Negative Positive 17 44/M 6 months 3.0 24 mg/dl; 3 cell/μl 0.46 Absent Positive Positive

[0106] TABLE 4 Age Immuno- CSF Protein Patient Age/ of modulatoryConcentration; CSF CSF Ig Oligoclonal CSF CSF CSF Ig vs EBs by NumberSex Onset EDSS Drugs Cell count Index Bands Culture PCR/S Western Blot 135/M 30 4.0 None 53 mg/dl;  2 cells/μl 1.07 Present Positive PositivePositive 2 51/M 31 7.5 None 69 mg/dl;  0 cells/μl 0.5 Present PositivePositive Positive 3 40/M 38 6.5 None 85 mg/dl;  4 cells/μl 0.64 PresentNegative Positive Positive 4 42/F 38 6.0 IFN 1 alpha 18 mg/dl;  3cells/μl 1.73 Present Negative Positive Weakly Positive 5 42/F 33 7.0None 37 mg/dl;  2 cells/μl 2.69 Present Positive Positive Positive 647/M 33 7.0 None 112 mg/dl;  2 cells/μl 0.58 Present Positive PositivePositive 7 35/F 29 7.0 None 48 mg/dl;  2 cells/μl 3.69 Present PositivePositive Positive 8 31/M 25 5.0 None 41 mg/dl;  1 cells/μl 0.5 AbsentPositive Positive Weakly Positive 9 50/M 40 6.5 MTX 24 mg/dl;  3cells/μl 0.6 Present Positive Negative Weakly Positive 10 37/F 34 3.5None 34 mg/dl; 15 cells/μl 0.77 Present Present Positive Weakly Positive11 54/M 44 8.5 MTX 82 mg/dl; 15 cells/μl 1.39 Present Positive PositivePositive 12 29/F 21 8.0 IFN 1 beta 14 mg/dl;  1 cells/μl 0.8 PresentPositive Positive Weakly Positive 13 44/F 23 7.5 None 59 mg/dl;  1cells/μl 0.7 Present Positive Positive Positive 14 36/M 32 4.5 MTX 31mg/dl;  0 cells/μl 0.52 Absent Negative Positive Negative 15 42/F 38 3.5None 32 mg/dl;  0 cells/μl 0.6 Present Positive Positive Weakly Positive16 54/F 40 6.5 None 54 mg/dl;  4 cells/μl 1.4 Present Negative PositivePositive 17 43/M 21 6.0 IFN 1 beta 54 mg/dl;  4 cells/μl ND PresentPositive Positive Positive 18 28/M 18 4.5 IFN 1 beta 33 mg/dl;  2cells/μl 1.2 Present Present Positive Positive 19 48/F 24 8.5 None 48mg/dl;  5 cells/μl 0.7 Present Positive Positive Positive 20 34/M 32 6.0None 115 mg/dl; 30 cells/μl 0.5 Present Positive Positive Positive

[0107] TABLE 5 CSF Protein Patient Age/ Neurologic Concentration; CSFCSF Ig Oligoclonal CSF CSF CSF Ig vs EBs by Number Sex Diagnosis Cellcount Index Bands Culture PCR/S Western Blot 1 44/F AIDP 121 mg/dl;  0cells/μl 0.2 Absent Negative Positive Weakly Positive 2 65/M CNSWegener's 35 mg/dl;  2 cells/μl 0.5 Absent Negative Negative Negative 319/F Encephalitis 18 mg/dl;  15 cells/μl ND Absent Negative NegativeNegative 4 32/M CNS Vasculitis 121 mg/dl;  0 cells/μl 2.6 AbsentNegative Negative Negative 5 36/F Paraneoplastic 104 mg/dl;  13 cells/μl0.5 Present Negative Negative Weakly Positive Encephalitis 6 25/MChronic 155 mg/dl; 227 cells/μl 0.5 Present Negative Negative NegativeMeningitis 7 41/F Granulomatous 88 mg/dl;  10 cells/μl 0.5 AbsentNegative Negative Negative Angitis 8 32/F PIE 95 mg/dl; 103 cells/μl0.47 Absent Positive Negative Weakly Positive 9 39/F Polyneuropathy 54mg/dl;  0 cells/μl 0.5 Absent Negative Negative Negative 10 59/F CNSVenous 72 mg/dl;  7 cells/μl 0.55 Absent Negative Negative NegativeThrombosis 11 66/F Brain Abscess 31 mg/dl;  2 cells/μl 0.37 AbsentNegative Negative Negative 12 28/M CNS Vasculitis 30 mg/dl;  8 cells/μl0.4 Absent Negative Negative Negative 13 24/F CNS Vasculitis 53 mg/dl; 0 cells/μl 0.4 Absent Negative Negative Negative 14 80/F CNS Wegener's63 mg/dl;  11 cells/μl ND ND Negative Negative Negative 15 44/MHydrocephalus 146 mg/dl;  14 cells/μl 0.38 Absent Negative NegativeNegative 16 38/F HSV-2 Myelitis 74 mg/dl;  4 cells/ml 0.2 ND NegativeNegative Negative 17 50/F Encephalopathy 25 mg/dl;  2 cells/ml ND AbsentNegative Negative Weakly Positive 18 34/M Thoracic 59 mg/dl;  21cells/μl 0.48 Absent Negative Positive Negative Myelitis 19 36/F BrainTumor 137 mg/dl;  6 cells/μl 0.48 Absent Negative Positive Negative 2054/M Stroke 44 mg/dl;  0 cells/μl ND ND Negative Negative Negative 2152/F Myelitis 51 mg/dl;  2 cells/ml 0.50 Absent Positive PositiveNegative 22 36/F Aseptic 39 mg/dl;  13 cells/ml 0.38 Absent NegativeNegative Negative Meningitis 23 41/F HSV-2 Myelitis 58 mg/dl;  0cells/μl 0.54 Absent Positive Positive Negative 24 36/F CNS Lupus 57mg/dl;  0 cells/μl 0.46 Absent Negative Negative Negative 25 28/FVasculitis 58 mg/dl;  1 cells/μl 0.49 Absent Negative Negative Negative26 38/M Lumbrosacral 62 mg/dl;  0 cells/μl 0.47 Absent Negative NegativeNegative Plexopathy 27 62/M W-K 50 mg/dl;  0 cells/μl ND Absent NegativeNegative ND Encephalopathy

[0108] Culture of C. pneumoniae from CSF

[0109] To at least 300 μl of a recently collected CSF sample, 200 μl oftrypsin (0.25%) ethylenediaminetetraacetic acid (1 mM) (EDTA; GIBCO BRL,Gaithersburg, Md.) in Hank's balanced salt solution (HBSS; GIBCO) at pH7.2 was added to achieve a final concentration of 0.1% trypsin; thesample was vortexed and then incubated at 37° C. for 30 minutes.Following incubation, the sample was again vortexed, centrifuged for 45minutes at 12,000×g in a microcentrifuge, and the pellet resuspended in1 ml of Iscoves medium (GIBCO); 0.5 ml of this diluted CSF sample wasadded to HL indicator cells (Human Lung Carcinoma Cells, WashingtonResearch Foundation, Seattle, Wash.) in each of two shell vials. Priorto adding the CSF sample, it is preferable that indicator HL cells bedemonstrated to be free of cryptic infection by C. pneumoniae. HL cellswere established as confluent monolayers on 12 mm cover slips, washedwith HBSS four times, treated with diethylaminoethyl-dextran (30 μg/ml)(DEAE-Dextran; GIBCO) in HBSS for 15 minutes, and washed again fourtimes with HBSS. After the CSF sample was added, the shell vials werecentrifuged at 4° C. at 1,800×g for one hour. To the spun shell vialswas added 1 ml of Iscoves or RPMI medium (GIBCO) containing 4 μg/mlcyclohexamide, 20% fetal calf serum (FCS; Hyclone, Logan, Utah)demonstrated to be free of C. pneumoniae and EBs, 4 mM L-glutamine(Sigma, St Louis, Mo.), and 100 μg/ml gentamicin (Sigma); the vials werethen incubated at 35° C. for seven days with additional centrifugation(4° C. at 1,800×g for 1 hour) on days 4, 5, and 6. Continuouspropagation for 14 days was achieved by a single culture passage afterseven days, followed by a second incubation period of seven days. Onecover slip of the duplicate shell vials was examined for C. pneumoniaeinclusions after each incubation period. Following fixation, the cellmonolayer was stained with a C. pneumoniae-specificfluorescene-conjugated mouse monoclonal antibody (1:50 dilution;Washington Research Foundation) and Evan's blue (3 μl/ml) in HBSScontaining 1% BSA, 0.15% Tween 20, or by an analogous immunocytochemicalstaining method. Enumeration of HL cells containing C. pneumoniaeinclusions was done under epi-fluoroscence using a Nikon Diaphot-TMDmicroscope with a B filter cassette. In the presence of Evans bluecounter stain, the emission spectrum was shifted toward the infrared forthis particular monoclonal antibody. Alternatively, a regular lightmicroscope may be used for inclusion bodies stained byimmunocytochemical methods.

[0110] For passage, the remaining vial was sonicated to remove the cellsfrom the cover slip after which EDTA to 1 mM final concentration wasadded. This vial was incubated at 37° C. for 30 minutes, centrifuged atlow speed (600×g for 5 min) to remove cell debris, and the supernatantwas centrifuged at 12,000×g for 45 min at ambient temperature. Thepellet was resuspended in 1 ml of Iscoves and 0.5 ml used to inoculateeach of duplicate fresh DEAE-Dextran-treated monolayers. This subculturewas centrifuged at 4° C. at 1,800×g for one hour at the time ofsubculture as well as again on days 4, 5, and 6. As a quality controlmeasure for this and all other laboratory methods, cell lines, FCS,media, or reagents of any type must be determined to be C.pneumoniae-free by PCR/Southern hybridization assay. All manipulationsof CSF samples and/or shell vials in which contamination might occurwere done in laminar-flow hoods (BL3) under continuous ultravioletlight.

[0111] PCR Amplification of Genes from C. pneumoniae

[0112] The MOMP Gene

[0113] To at least 300 μl of CSF sample, 200 μl of HBSS containing 0.25%trypsin and 1 mM EDTA at pH 7.2 was added to achieve a finalconcentration of 0.1% trypsin, and the sample vortexed and thenincubated at 37° C. for 30 minutes. Following incubation, the sample wasagain vortexed, centrifuged for 45 minutes at 12,000×g in amicrocentrifuge, and the pellet resuspended in 20 μl of lysis buffer(0.5% sodium dodecylsulfate (SDS), 1% NP40, 0.2 M NaCl, 10 μM DTT, 10 mMEDTA, 20 mM Tris-HCl at pH 7.5). To this was added 8 μl of proteinase K(20 μg/ml; Boehringer-Mannheim, Indianapolis, Ind.), after which thespecimen was mixed and incubated overnight at 37° C. From this specimen,purified DNA was extracted from the aqueous fraction with Na acetate(1:10 dilution by volume of a 3 M solution; Fisher Scientific,Pittsburgh, Pa.) and mixing/precipitation with 2:2.5 dilution by volumeof cold absolute ethanol after performing initial extraction with amixture of phenol:chloroform:isoamyl alcohol (25:24:1; Sigma Chemical,St. Louis, Mo.) followed by two extractions with chloroform. The DNA waswashed with 70% ethanol in water, spun (600×g for 5 minutes at ambienttemperature), and resuspended in 20 μl of water.

[0114] PCR was carried out using the entire MOMP gene (1.2 kb) usingDeep Vent polymerase in the manufacturer's buffer (New England Biolabs,Boston, Mass.) with no additional MgCl₂. The MOMP primers were asfollows. MOMP forward: ATG AAA AAA CTC TTA AAG TCG GCG TTA TTA TCC GCCGC (SEQ ID NO: 1); MOMP reverse: TTA GAA TCT GAA CTG ACC AGA TAC GTG AGCAGC TCT CTC G (SEQ ID NO: 2). Reaction mixtures contained 20 μl oftarget DNA, 200 picoMoles each primer, 200 μM each dNTP, and 1 unit ofDeep Vent polymerase. The PCR reaction was carried out for 35 cycles at94° C. for 1 minutes, 58° C. for 2 minutes, and 74° C. for 3 minutes.PCR reactions were analyzed by electrophoresis in 1% agarose gel for 45minutes at 95 volts. For confirmation and enhanced sensitivity, the gelswere further analyzed by Southern hybridization using adigoxigenin-labeled (DIG, Boehringer-Mannheim) MOMP gene probe from theTWAR strain of C. pneumoniae (ATCC VR-1310). Briefly, agarose gels weretreated with 0.4 M NaOH for 15 minutes, and the DNA transferred bycapillary blotting to positively charged nylon membranes(Boehringer-Mannheim). The TWAR MOMP gene was labeled with DIG(Boehringer-Mannheim). Membranes were prehybridized in hybridizationbuffer (10% Dextran sulfate, 1M NaCl, 1% SDS) for at least one hour at65° C. DIG-labeled probe (100 ng) was added in fresh hybridizationbuffer and incubated at 65° C. overnight. Blots were washed 3 times in2× SSC, 1% SDS at ambient temperature, and an additional three times at65° C. Final high stringency washes were performed in 0.1× SSC, 0.1% SDSat ambient temperature. Membranes were blocked in 5% w/v dehydratednon-fat milk in PBS, 0.2% Tween 20 for one hour at ambient temperature,then incubated in anti-DIG-alkaline phosphatase conjugated Fab fragments(Boehringer-Mannheim) diluted 1:5000 in PBS, 0.2% Tween 20, anddeveloped with NBT/BCIP substrate.

[0115] Nested PCR primers were chosen within the MOMP which are C.pneumoniae specific (located in variable domains 1 and 4) and yield a727 bp band. These primers are as follows. Nest MOMP forward: GCT GCTGCA AAC TAT ACT ACT GCC (SEQ ID NO: 3); Nest MOMP reverse: GAA TCA GTAGTA GAC AAT GCT GTG G (SEQ ID NO:4). The target for the nested primerswas 5:1 of PCR product from the full length MOMP reaction. The nestedPCR reaction was carried out under the same reaction conditions used forthe full length MOMP: 35 cycles at 94° C. for 1 minute, 45° C. for 2minutes, and 72° C. for 3 minutes, including a 7 minute extension at 72°C. at the end of the program. PCR reactions were analyzed byelectrophoresis in 1% agarose gel for 45 minutes at 95 volts. Forconfirmation and enhanced sensitivity of the nested PCR, the nested PCRgels were further analyzed by Southern hybridization using a DIG-labeledMOMP probe from the TWAR strain of C. pneumoniae.

[0116] Measurement of Antibody Levels in CSF to Whole Elementary BodyAntigens of C. pneumoniae by ELISA.

[0117] CSF antibodies to C. pneumoniae were measured by an ELISAtechnique as follows. 96-well plates were coated with 100 μl of C.pneumoniae EB antigens (250 ng/well). EB antigens were prepared fromconcentrated C. pneumoniae EBs by treating them with 25 mM DTT, 2%2-mercaptoethanol, and 2% SDS for five minutes at 100° C. Treated EBswere sonicated, centrifuged (500×g for 30 min at room temperature) andresuspended (2 μg/ml protein) in PBS pH 7.4. Concentrated C. pneumoniaeEBs were obtained by growing C. pneumoniae ATTC VR-1310 or otherisolates in 25 ml flasks containing a confluent growth of HL cells(Washington University Foundation) in Dulbecco's minimal essentialmedium (DMEM; GIBCO) and 10% FCS. Infection was facilitated by twowashes with HBSS followed by a 20 minute preincubation with DEAE-Dextran(30 μg/ml) in HBSS. The infectious inoculum was added in 2 ml volume ofserum-free media and the flasks centrifuged at 1,200×g for one hour at4° C. To the spun flasks was added 2 ml of Iscoves medium containing 4μg/ml cyclohexamide, 20% FCS, 4 mM L-glutamine, and 100 μg/mlgentamicin. The flasks were then incubated at 35° C. for three days atwhich time the infected cells began to lyse and release EBs. On day 4,the culture flasks were sonicated for 20 seconds, and the cell debrisremoved by centrifugation at 600×g for five minutes at room temperature.The supernatant containing the infectious EBs was centrifuged at18,000×g for 30 minutes and the pellet resolubilized in water containing25 mM DTT, 2% 2-mercaptoethanol, and 2% SDS. After the EB antigens wereadded to the 96-well plate, the plates were incubated overnight and thenunoccupied sites in the wells blocked with 1% BSA, PBS-Tween 20 for onehour. The CSF samples were added to each well in a final concentrationof 1 μg of immunoglobulin diluted in 100 μl of PBS as determined by ratelaser nephelometric methods (Behring Nephelometer Analyzer II, BehringDiagnostics Inc, San Jose, Calif.) and incubated overnight at 4° C.Following this step, the plates were washed with PBS-Tween 20, and thenperoxidase-conjugated goat anti-human IgG (1:10,000) (Sigma) was added.Following two hours of incubation, the plates were washed with PBS-Tween20 and the substrate added. The absorbance units at 405 nm were readusing an ELISA reader (Bio-Tech Instruments, Burlington, Vt.) at 1 hour.

[0118] Western Blot Assays of Elementary Body Antigens Reactive AgainstCSF Immunoglobulins

[0119] EB antigens were prepared from concentrated C. pneumoniae EBs bytreating them with 25 mM DTT, 2% 2-mercaptoethanol, and 2% SDS for 5minutes at 100° C. as done for the ELISA procedure. Treated EBs weresonicated, and then 2.5 μg of the sonicated protein was loaded in eachwell and run on an 8% SDS-PAGE gel at 100V for two hours at ambienttemperature. The gel was transferred to nitrocellulose membrane at 100Vfor one hour at ambient temperature. Individual strips were cut andincubated in 3% BSA-tris-buffered saline with Tween 20 (TBST) for twohours at ambient temperature to block unoccupied sites. The strips werethen washed three times with TBST, and finally incubated with CSF(containing 5 μg of immunoglobulin) for two days at 4° C. Followingincubation with CSF, the strips were washed three times with TBST andincubated with peroxidase-conjugated goat anti-human IgG (1:500) (Sigma)for 1.5 hours at ambient temperature and examined using achemiluminiscent detection assay (Pierce, Rockford, Ill.). Controls usecell lysates of uninfected HL cells instead of C. pneumoniae EBs inWestern blot assays.

[0120] B) Results

[0121] Detection of C. pneumoniae in CSF

[0122] Direct evidence for the presence of C. pneumoniae in the CNS wasdetermined by CSF cultures. The majority of cultures were read aspositive after the second passage. Cultivation and isolation of C.pneumoniae was performed according to a rigorous protocol designed tomaximize yield. Among patients with newly diagnosed relapsing remittingMS, 47% of patients (8/17) had C. pneumoniae isolated from CSF cultures(Table 3). Among patients with progressive MS, 80% of patients (16/20)were culture positive (Table 4). One culture-negative MS patient (#3)was taking ofloxacin for a urinary tract infection at the time of CSFculture. C. pneumoniae was isolated from CSF in 3 OND control patients(Table 5). One of these three patients (#8) was diagnosed as having postinfectious encephalomyelitis (PIE), which may, in fact, represent avariant of MS. The remaining two patients presented with inflammatorymyelopathy; one case of unknown etiology and the other case thought tobe due to HSV-2. In the latter two patients, changes consistent withinflammatory myelopathy were seen on MRIs of the spinal cords.

[0123] The presence of C. pneumoniae in the CNS was also evaluated bypolymerase chain reaction (PCR) methods which assayed CSF for the majorouter membrane protein (MOMP) gene of C. pneumoniae. The specific 1.2 kbband for the MOMP gene seen following ethidium bromide staining ofagarose gels was confirmed by Southern hybridization using labeled MOMPgene probes (Dalhoff and Maass, Chest 110:351-356, 1996). The MOMP genefor C. pneumoniae was amplified and confirmed in all 17 (100%) relapsingremitting MS patients (Table 3) and 19 of 20 (95%) progressive MSpatients (Table 4) versus 5 of 27 (18%) OND controls (Table 5). Oneprogressive MS patient (#9) and one OND control (#8) were negative forthe MOMP gene, but were positive by culture. Of the five OND controlpatients who were positive for the MOMP gene, three had thoracicmyelitis (#18, #21, #23), the fourth (#1) had acute inflammatorydemyelinating polyneuropathy (AIDP), while the fifth (#20) had a stroke.In both groups of relapsing remitting and progressive MS patients, allculture-negative MS patients were positive by PCR/Southern hybridization(PCR/S) assays.

[0124] Indirect evidence of C. pneumoniae infection in the CNS wasdetermined by the detection of CSF antibodies against preparations of C.pneumoniae elementary bodies that had been reduced, solubilized, andsonicated using an ELISA method (Ladany et al., J. Clin. Microbiol.27:2778-2783, 1989). To ensure that differences in the ELISA absorbancesignal were not due to differences in the concentration of antibodies inthe CSF, the amount of immunoglobulins in CSF was determined bynephelometric methods in order to add equal amounts to the ELISA plates.Mean absorbance OD values for anti IgG antibody response to EB antigensusing CSF from 17 relapsing remitting MS patients was 0.185±0.042 whilethe mean OD in the control OND group was 0.078±0.025 (p<0.01 Fisher'stest). Of 17 patients with relapsing remitting MS, 15 patients (88%) hadOD values that were three standard deviations from the OND group. Theanti IgM response to EB antigens of C. pneumoniae expressed as OD unitswas 0.115±0.02 in the RRMS group and 0.086±0.011 in the OND group(p<0.05). When the antibody titers in patients with progressive MS wereexamined, the mean OD of 20 progressive MS patients was 0.237±0.11,while in the OND control group it was 0.093⁺0.022 (p<0.001 Fisher'stest). Seventeen of 20 (85%) MS patients tested had absorbance values inCSF that were three standard deviations greater than those seen incontrols. These observations demonstrated that increased CSF antibodiesagainst C. pneumoniae were present in the majority of patients withrelapsing-remitting and progressive MS.

[0125] The specificity of the CSF antibodies was evaluated by Westernblot assays using EB antigens (Friedank et al., Eur. J. Microbiol.Infect. Dis. 12:947-951, 1993). Equal amounts of CSF immunoglobulinswere incubated with EB antigens in order to control for differences inimmunoglobulin concentrations in the CSF. All relapsing remitting MSpatients (17/17) showed prominent reactivity to a 75 kD protein of C.pneumoniae. In addition, 17 of 20 CSF samples from MS patientsdemonstrated prominent reactivity to a 75 kD protein of C. pneumoniaewith weaker reactivity to 65 kD, 60 kD, and 55 kD proteins observed in13 of these 20 MS patients. In 19 of these 20 MS patients, the strengthof the bands seen on Western blot correlated with the ELISA OD units. Incontrast, reactivity to this 75 kD protein was seen in only 4 of 20 ONDcontrols. In all 4, the reactivity was weak when compared to MSpatients. One of these OND control with PIE (#8) was culture-positivefor C. pneumoniae. Another patient (#1) with AIDP was positive by PCR/Sto C. pneumoniae but culture-negative. The other two OND controls (#5and #17) with positive Western blots were negative for C. pneumoniae byculture and PCR/S (Table 5).

[0126] The pattern of antibody reactivity on Western blots was similarin all MS patients. The nature of the 75 kD protein band seen in themajority of MS patients as well as in three OND controls is not known.Silver-stained gels of C. pneumoniae EB antigens failed to show adominant band at that molecular weight. Others have reported antibodyreactivity to a 75 kD heat shock protein in the serum of patientsfollowing C. pneumoniae infection (Campbell et al., Infect. Immun.57:71-75, 1989). When Western blots were performed using cytosoliclysates from uninfected HL cells, no binding of antibody was seen ineither the MS patient or the OND group, demonstrating that the antibodybinding was specific for elementary body-antigens of C. pneumoniae.

[0127] MS patients are known to have an increase in CSF immunoglobulinsin which a portion of this increase is seen as oligoclonal bands onisoelectric focusing gels. These oligoclonal bands represent cationicantibodies that have isoelectric points in the anodic region of the gel.The presence of these cationic antibodies in the CSF was evaluated byisoelectric focusing (IEF) of CSF followed by Western blot assays usingEB antigens. Of 20 progressive MS patients, 12 had CSF immunoglobulinsat isoelectric points of 7.5 or greater, that reacted with EB antigens.Two OND control patients, one of whom was positive by culture (#8) andthe other by PCR/S (#1) demonstrated similar cationic antibodies againstEB antigens. These results suggest that cationic anti-chlamydialantibodies are present in the CSF of patients with MS and represents, inpart, the specificities for the characteristic oligoclonal bands seen inMS. These and additional findings are described in Example 3, below.

EXAMPLE 2 Detection of the C. pneumoniae 16S RNA Gene in the CSF of MSPatients

[0128] A) Materials and Methods

[0129] Patients and Patient Selection

[0130] Seventeen patients with relapsing-remitting MS, six patients withprogressive disease (five secondary progressive, one primaryprogressive) who satisfied the Poser criteria for definite MS, wereselected for the study. Age and gender matched controls were recruitedfrom 13 patients with other neurologic diseases (OND) in whom CSF wasbeing obtained for diagnostic studies. In addition, CSF from twopatients with subacute sclerosing pan encephalitis were examined in theimmunoblot assays.

[0131] PCR Amplification of 16S rRNA Gene of C. pneumoniae

[0132] To at least 300 μl of CSF sample in its original collection tube,200 μl of HBSS containing 0.25% trypsin, 1 mM EDTA at pH 7.2 was addedto achieve a final concentration of 0.1% trypsin. The sample was thenvortex mixed and incubated at 37° C. for 30 minutes. Followingincubation, the sample was again vortex mixed and centrifuged for 45minutes at 12,000×g in a microcentrifuge at ambient temperature. Thepellet was resuspended in 20 μl of lysis buffer (0.5% SDS, 1% NP40, 0.2M NaCl, 10 μM DTT, 10 mM EDTA, and 20 mM Tris-HCl at pH 7.5). Followingperturbation of the chlamydial surface by reduction of its extensivedisulfide bonding, 8 μl of proteinase K (20 μg/ml) was added. Thespecimen was mixed and incubated at 37° C. overnight. Purified DNA wasthen extracted from the aqueous fraction of the specimen with Na acetate(1:10 dilution by volume of a 3 M solution) and mixing/precipitationwith 2:2.5 dilution by volume of absolute ethanol at 4° C. afterperforming initial extraction with a mixture ofphenol:chloroform:isoamyl alcohol (25:24:1; Sigma Chemical) followed bytwo extractions with chloroform. The DNA precipitate was washed with 70%ethanol in water, centrifuged at 600×g for five minutes at ambienttemperature, and resuspended in 20 μl of water.

[0133] Nested PCR was carried out to detect the 16S rRNA gene of C.pneumoniae as follows: The 16S rRNA gene primers used were outer forward(TTT AGT GGC GGA AGG GTT AGT A (SEQ ID NO: 5)), outer reverse (CAC ATATCT ACG CAT TTC ACC G (SEQ ID NO: 6)), inner forward (CTT TCG GTT GAGGAA GAG TTT ATG C (SEQ ID NO: 7)), and inner reverse (TCC TCT AGA AAGATA GTT TTA AAT GCT G (SEQ ID NO: 8)). With this nested PCR procedure,the outside 16S rRNA primers amplify members of the Chlamydia genuswhile the inner 16S rRNA primers are specific for C. pneumoniae andamplify a 446 base pair sequence. Reaction mixtures for the outer primerreaction contain 20 μl of target DNA, 200 pM of each outer primer, 200μM of each dNTP, and 1 unit of Deep Vent polymerase in the manufacturersbuffer (New England Biolabs) with no additional MgCl₂. The PCR reactionwas performed for 35 cycles at 94° C. for 1 minute, 55° C. for 2minutes, and 74° C. for 3 minutes. Five microliters of the reaction mixis removed from the reaction tube and placed in a second tube containingthe same components with the exception that inner primers are usedinstead of the outer primers. Reaction conditions for the second nestedphase were 35 cycles at 94° C. for 1 minute, 50° C. for 2 minutes, and74° C. for 3 minutes. The reaction products are then subjected toelectrophoresis in 1% agarose gels for 45 minutes at 95 V. Amplified DNAwas transferred from the agarose gel to positively charged nylonmembranes (Boehringer-Mannheim) by capillary blotting. The 16S rRNA genehomologous to the inner primers first was obtained by PCR from the TWARstrain of C. pneumoniae (VR-1310, ATCC). This inner primer product wasthen labeled with DIG following the manufacturers directions.DIG-labeled inner product was used as a probe for membranesprehybridized at 65° C. in hybridization buffer (10% dextran sulfate, 1M NaCl, 1% SDS) by adding 100 ng of DIG-labeled probe in freshhybridization buffer and incubated overnight at 65° C. overnight. Blotswere washed three times in 2× saline-sodium citrate containing 1% SDS atambient temperature and an additional three times at 65° C. followed byhigh stringency washes in 0.1× saline-sodium citrate containing 0.1% SDSat ambient temperature. Membranes were blocked in 5% w/v dehydratednonfat milk in PBS, 0.2% Tween 20 for one hour at ambient temperature,then incubated in anti-DIG-alkaline phosphatase-conjugated Fab fragments(Boehringer-Mannheim) diluted 1:5000 in PBS, 0.2% Tween 20, anddeveloped with NBT/BCIP substrate.

[0134] B) Results

[0135] Presence of 16S rRNA in CSF Correlates with MS

[0136] Fifteen of 17 (88%) of the CSF samples from MS patients containedDNA specific for the 16S rRNA gene of C. pneumoniae (FIG. 1). Of these15 CSF samples that were positive by PCR, 8 yielded strongly positivesignals for the 446 base pair C. pneumoniae product. Because the 643base pair outer primer product also was present in the sample used inthe nested PCR, labeling of it was seen as would be expected. One of theCSF samples from MS patients read as negative (number 5, lower gel)contained hybridization-specific products with a significantly highersize of the major band. This may represent a Chlamydia with a mutationwithin the inner primers yielding a product of approximately 520 basepairs. In contrast, only two of the 13 CSF samples from the OND controlgroup (number 3, upper gel and number 1, lower gel) yielded some weakhybridization product of the incorrect base pair size and were scored asPCR negative.

EXAMPLE 3 Cationic Anti-Chlamydial Antibodies in the CSF of Patientswith MS

[0137] To further demonstrate that C. pneumoniae infection is causal tothe development of MS, we analyzed the specificity of the intrathecalhumoral response and, in particular, the reactivity of the oligoclonalbands from patients with relapsing remitting and progressive MS againstC. pneumoniae antigens. In virtually every chronic infection of the CNS,increased levels of immunoglobulins that recognize the pathogen aresynthesized exclusively within the CNS compartment and are seen asoligoclonal bands by IEF methods. In MS, oligoclonal bands are ahallmark of the disease, although the antigenic specificity of thesebands has been unknown. Described below are experiments that examine thepattern and specificity of reactivity of oligoclonal bands (representingintrathecal antibody synthesis) to C. pneumoniae, MBP, measles, andHSV-1 antigens in MS patients and OND controls.

[0138] A) Materials and Methods

[0139] Patients and Patient Selection

[0140] Patients who satisfied the criteria of definite MS were recruitedfor the present study. In all, 15 MS patients (eight secondaryprogressive, two primary progressive, five relapsing remitting) werestudied. Age and gender matched OND patients in whom CSF was beingobtained for diagnostic studies, served as controls and have also beendescribed previously. In all patients, CSF and, when possible, serum wasaliquoted into 0.5 ml freezing vials and stored at −70° C. before use.CSF samples from patients with subacute sclerosing pan-encephalitis(SSPE) were a kind gift of Dr. ter Muelen (Freiberg, Germany). Dr. S.Jacobson (NIH, Bethesda, Md.) kindly provided CSF samples from patientswith HTLV- 1 myelopathy.

[0141] Preparation of Purified EBs of C. pneumoniae

[0142] EB antigens of C. pneumoniae were prepared from concentrated EBsby treating them with 25 mM DTT and 2% 2-mercaptoethanol for 5 minutesat 100° C. EBs were then sonicated and centrifuged (500×g for 30 minutesat room temperature). EB antigens were resuspended (20 μg/ml protein) inPBS pH 7.4 and used for all experiments. Concentrated C. pneumoniae EBswere obtained by growing C. pneumoniae (VR-1310; ATCC) in the HL cellline. EBs were harvested and resolubilized in Iscoves minimal essentialmedium and their purity assessed by SDS-PAGE electrophoresis followed byWestern blotting with anti-C. pneumoniae antibody (Accurate Chemical &Scientific Corp, Westbury, N.Y.)

[0143] Measurement of Antibody Levels in CSF to EB Antigens of C.pneumoniae by ELISA

[0144] Microtiter 96-well plates were coated with EB antigens of C.pneumoniae (1 μg/well). Plates were incubated overnight and unoccupiedsites blocked with 1% BSA/PBS-Tween 20 for one hour. The CSF sampleswere added to each well in a final concentration of 1 μg ofimmunoglobulin diluted in 100 μl of PBS as determined by nephelometricmethods and incubated overnight at 4° C. Following this step, the plateswere washed with PBS-Tween 20, and then peroxidase-conjugated goatanti-human IgG (1:10,000) (Sigma Chemical Co.) was added. Following twohours of incubation, the plates were washed with PBS-Tween 20 and thesubstrate added. The absorbance at 405 nm was read using an ELISA reader(BioTech Instruments, Burlington, Vt.) at one hour. CSF from fivepatients in whom all CSF studies were normal was pooled and served as aninternal control. This pooled CSF was used to determine basal opticaldensity units to EB antigens in the ELISA. Antibody titers to C.pneumoniae EBs were represented as an antibody index, defined as theratio of absorbance in OD units for the test patient divided by OD unitsof the control group.

[0145] Affinity-Driven Immunoblot Technique for Detection of AntigenicSpecificity of Oligoclonal Bands

[0146] To determine the antigenic specificity of antibodies in CSF, weperformed isoelectric focusing of CSF immunoglobulins, followed byaffinity-driven transfer of antibodies onto antigen-coated membraneswith 0.25 μg of immunoglobulin from CSF obtained from MS patients andOND controls using an Isoelectric Focus Units System (Wallac Inc, Akron,Ohio). Focusing was carried out in agarose gel (pH 3.0-10.0) accordingto the manufacturer's protocol. Capillary transfer was used to transferthe IEF gel to nitrocellulose paper (Trans-blot, 0.45 μm; Bio-Rad, SanFrancisco, Calif.) that was pre-coated with antigen. The membrane waspre-coated with C. pneumoniae EB or viral antigens at a concentration of5 μg/ml and incubated overnight with gentle rocking at 4° C. Controlantigens for blotting experiments were measles, HSV-1 (Bio-Whittaker,Inc, Walkersville, Md.), and guinea pig MBP. Unoccupied sites wereblocked with 5% fat-free milk. Antibody bound to antigen was probed withperoxidase-conjugated goat anti-human IgG (1:10,000) (Sigma) using achemiluminescent detection assay.

[0147] Solid Phase Adsorption of Cationic Antibodies in CSF to C.pneumoniae

[0148] Purified EBs of C. pneumoniae were heated to 100° C. for fiveminutes, sonicated for 30 seconds, resuspended in carbonate buffer, pH9.6, and coated (20 μg/well) overnight onto microtiter 96 well plates.The wells were washed in PBS and unoccupied sites blocked using 1% BSAfor two hours. CSF samples containing 0.8 μg of Ig in 200 μl of saline(pH 7.4) were added to the 96 well plates. Control antigens (20 μg/well)to which CSF Ig samples also were incubated included MBP, measles, andHSV-1. After overnight incubation at 4° C., CSF containing unbound Igwas carefully removed and lyophilized. Immunoglobulins were redissolvedin 30 μl of water immediately prior to running an IEF gel. Samplescontaining 0.25 μg of Ig (10 μl) were loaded into an IEF gel, andisoelectric focusing was performed. The gel was transferred ontonitrocellulose membranes, and the presence of Ig bound to antigen on themembranes was probed with peroxidase-conjugated goat anti-human IgG(Sigma) using a chemiluminescence detection assay (Amersham, ArlingtonHeights, Ill.).

[0149] B) Results

[0150] Measurement of Antibodies (IgG and IgM) in CSF of MS Patients andOND Controls against EB Antigens of C. pneumoniae by ELISA Methodology

[0151] Antibody titers to C. pneumoniae were measured using ELISAmethodology for 10 patients with progressive (secondary and primary) MS,five patients with relapsing remitting MS, and 14 OND controls. The meananti-IgG antibody index to C. pneumoniae antigens in the 15 MS patientswas 6.1±2.9 (p<0.001 versus OND control; FIG. 1). In all but three MSpatients, the antibody index was at least three standard deviationsgreater than that seen in controls. The mean antibody index in the 12OND control patients was 1.3±0.8. In the two SSPE patients, the antibodyindex was 2.5 and 2.6, while in the remaining 10 OND patients, theantibody index ranged between 0.5 and 2.2. The mean anti-IgM antibodyindices to C. pneumoniae in MS patients and controls were notstatistically different (antibody index for MS was 2.0±1.6, OND1.3±0.5). These results reflect the persistence of the intrathecalhumoral immune response to C. pneumoniae in MS patients over that of ONDcontrols.

[0152] Affinity-Driven Immunoblots for Antigen Specificity ofOligoclonal Bands in MS Patients and OND Controls

[0153] In all 15 MS patients, cationic antibodies (seen as oligoclonalbands) showed binding to C. pneumoniae antigens followingaffinity-driven immunoblot transfer (Table 6). Representative patternsof the immunoblots following transfer onto antigen-coated membranes areshown in FIG. 2 (four MS patients) and FIGS. 3A-3D (four OND controls).In all MS patients, the binding of C. pneumoniae antigens to thecathodal antibodies closely reflected the CSF immunoglobulin patternseen on IEF gels transferred onto non-antigen-coated membranes. Theprominence of the signal of individual oligoclonal bands followingtransfer onto EB-coated membranes differed in intensity, although not intheir overall pattern, suggesting differences in the affinity of theindividual oligoclonal bands to the antigen. When reactivity to otherantigens were examined, weak binding to measles antigen was seen in nineof 15 patients (see, for example, FIG. 3A and Table 6). None of thesebands matched patterns to that seen following transfer of immunoglobulinto C. pneumoniae-coated membranes. Representative patterns of fouraffinity-driven immunoblots for OND controls are shown in FIGS. 4A-4D.In two SSPE patients, as expected, oligoclonal bands seen on IEF gelswere bound to measles antigen following transfer. In patient #2 (Table7), reactivity of some of the cathodal antibodies to C. pneumoniaeantigens was seen. Weak binding to C. pneumoniae antigens was also seenfor patient #9 (Table 7), who presented with clinical features of HSV-2myelitis. This patient was positive in the CSF by PCR and culture for C.pneumoniae. This patient has now presented with a second lesion in thethoracic cord and fulfills the clinical criteria for relapsing remittingMS. TABLE 6 EDSS Oligoclonal ELISA ELISA Immunoblot ImmunoblotImmunoblot Adsorption Adsorption Patient Age/Sex Scale IgG Bands IgG IgMMeasles HSV-1 C. pneumoniae EB Ag Measles 1 40/M 6.5 0.64 Positive 4.21.89 Positive Negative Positive Yes/Partial No 2 54/M 8.5 1.38 Positive14.0 2.0 Weak Positive Negative Positive Yes ND 3 42/F 6.5 1.73 Positive6.2 5.0 Positive Positive Positive Yes No 4 36/F 8.0 NA Positive 6.2 2.2Negative Negative Positive Yes No 5 35/F 7.5 3.69 Positive 9.25 1.6Positive Positive Positive Yes No 6 29/M 3.0 1.2 Positive 6.0 0.7Positive Negative Positive Yes No 7 28/M 3.5 2.3 Positive 7.25 1.55Negative Positive Positive Yes/Partial No 8 55/M 8.5 NA Positive 8.6 1.4Positive Negative Positive Yes No 9 51/M 7.0 0.49 Positive 3.0 1.5Positive Negative Positive Yes ND 10 46/F 7.0 0.9 Positive 7.6 NDNegative Negative Positive No No 11 44/M 3.5 0.67 Positive 3.2 1.6Negative ND Positive No No 12 20/F 1.0 1.4 Positive 2.0 1.1 Positive NDPositive Yes No 13 24/F 6.5 0.9 Positive 5.2 1.5 Negative ND PositiveYes No 14 49/M 1.5 1.19 Positive 5.0 2.0 Negative ND PositiveYes/Partial No 15 26/F 3.0 1.09 Positive 5.7 4.2 Positive ND PositiveYes No

[0154] TABLE 7 Oligoclonal ELISA ELISA Immunoblot Immunoblot AbsorptionAbsorption Patient Age/Sex Diagnosis Bands IgG IgM Measles C. pneumoniaeC. pneumoniae Measles 1 NA SSPE Positive 2.6 1.5 Positive Negative NoYes 2 NA SSPE Postive  2.5 1.2 Positive Positive No Yes 3 NA SSPEPositive ND ND Positive Negative No Yes 4 32/M Vasculitis Negative 1.72.0 Positive Negative ND ND 5 36/F CNS Lupus Negative 0.9 0.5 PositiveNegative No ND 6 26/M Meningitis Positive 0.5 1.1 Negative Negative NoNo 7 52/M CNS Syphilis Positive 1.35 2.0 Positive Negative No No 8 38/FHSV-2 Negative ND ND Positive Negative ND ND Myelitis 9 28/F HSV-2Negative 1.25 1.5 Negative Weak Positive No No Myelitis 10 36/M CNSSarcoid Negative 0.75 0.9 Negative Negative No No 11 69/F VasculitisNegative 1.56 1.5 Positive Negative ND ND 12 NA HTLV-1 NA 0.95 2.09Negative Negative No No Myelitis 13 NA HTLV-1 NA 2.25 1.47 NegativeNegative ND ND Myelitis 14 NA HTLV-1 NA 1.66 1.86 Negative Negative NDND Myelitis

[0155] Solid Phase Adsorption of Oligoclonal Bands with EB Antigens ofC. pneumoniae

[0156] If oligoclonal bands represent the dominant CNS humoral responseto C. pneumoniae infection, we predicted the adsorption of these bandsby antigens of C. pneumoniae. Adsorption was carried out in solid phasewith a 25-fold excess of antigen over antibody (0.8 μg of CSF IgG platedonto microtiter wells incubated with 20 μg/well of antigen). In parallelexperiments, CSF samples containing 0.8 μg of IgG were added to wellscoated with 25-fold excess of MBP, measles, or HSV- 1 antigens, whichserved as antigen specificity controls. In 10 MS patients, theadsorption by C. pneumoniae EB antigens was complete as no oligoclonalbands were seen at isoelectric points greater than 7.5 (FIGS. 5A-5J). Inthree of the remaining five MS patients, adsorption of oligoclonal bandswith C. pneumoniae was incomplete (FIGS. 6A-6C), while, in two, no clearevidence of adsorption was seen (FIGS. 6D and 6E; Table 6, patients #10and 11).

[0157] Nine patients in the OND group were studied; representativepatterns of six are shown in FIGS. 7A-7F. No changes in thechemiluminescence signal of the oligoclonal bands were seen followingadsorption with C. pneumoniae antigens in eight of nine patients.Oligoclonal bands were adsorbed with excess measles antigen in all threeSSPE patients, but not with HSV- 1 or EB antigens, suggesting that theanti-measles antibody response in the CSF constituted the major antibodyresponse in SSPE patients. In patient #2 (Table 7), cathodal antibodiesreactive to EB antigens of C. pneumoniae were seen on affinity-drivenimmunoblots (FIG. 3A-3D). Incubation of CSF from SSPE-2 with EB antigensof C. pneumoniae did not alter the IEF gel, suggesting that the anti-C.pneumoniae antibodies did not comprise the major antibody response inthe CSF. In the remaining five patients with inflammatory disease of theCNS, no difference in the banding pattern of cathodal antibodies wasseen following adsorption with EB antigens of C. pneumoniae. Theseresults suggest that the majority of oligoclonal bands in CSF of MSpatients represent antibodies to C. pneumoniae antigens. Non-specificadsorption of antibodies to C. pneumoniae antigens in MS patients was anunlikely explanation, since antibodies present in the anodal region didnot bind to C. pneumoniae antigens. Also, no decrease in the oligoclonalbands was seen among nine OND controls following incubation with C.pneumoniae antigens.

EXAMPLE 4 Beta Interferon Enhances Intracellular Nitric Oxide Activityand Inhibits Secretion of Interleukin-12/p40

[0158] A) Materials and Methods

[0159] Mice and Reagents

[0160] Female SJL/J mice from Clarence Reader (National Institutes ofHealth, Bethesda, Md.) were maintained in the animal care facility atVanderbilt University Medical Center. Murine β-IFN and sheepanti-mouse-β-IFN antibodies were obtained commercially (Bio-SourceInternational, Camarillo Calif.). Control sheep immunoglobulin and LPSwere obtained from Sigma Chemical (St. Louis, Mo.). Monoclonal ratanti-murine IL-12 hybridomas C17.15 and C15.8 were supplied by G.Trinchieri (Wistar Institute, Philadelphia, Pa.), and the respectiveantibodies were purified from ascitic fluid from nude mice.

[0161] Preparation and Stimulation of Splenic Macrophages

[0162] Splenocytes were washed twice in PBS and plated in 24-wellculture plates (Corning, Corning, N.Y.) in Dulbecco's minimal essentialmedium (DMEM; GIBCO BRL, Gaithersburg, Md.) containing 10% fetal calfserum (FCS; Hyclone, Logan, Utah) at 37° C. for two hours. Non-adherentcells were washed away by gentle rinsing in warm medium, and themacrophages were allowed to adhere to the plastic wells overnight. Foroptimal growth of macrophages, colony stimulating factor-1 (CSF-1)obtained from culture supernatants of LADMAC cells (American Type TissueCollection (ATCC), Manassas, Va.) was added to reach a finalconcentration of 20%. After reaching confluence, cells were removed fromthe plastic wells by gentle trituration and examined for their purity bystaining with fluoroscein-conjugated anti-CD11b and anti-I-As (clone10-3.6) antibodies. These studies indicated that greater than 90% ofcells expressed the macrophage phenotype.

[0163] Preparation of EB Antigens

[0164] EB antigens were prepared from concentrated C. pneumoniae EBs bytreating them with 25 mM DTT, 2% 2-mercaptoethanol, and 2%dodecylsulfate (SDS) for 5 minutes at 100° C. Treated EBs weresonicated, centrifuged (500×g for 30 minutes at room temperature) andthe supernatant resuspended at 2 μg/ml protein in PBS pH 7.4.Concentrated C. pneumoniae EBs were obtained by growing C. pneumoniae(VR-1310; ATTC) in 25 ml flasks containing a confluent growth of HLcells (Human Lung Carcinoma Cells; Washington University Foundation,Seattle, Wash.) in DMEM and 10% FCS . Infection was facilitated by twowashes with Hank's balanced salt solution (HBSS; GIBCO) followed by a 20minute pre-incubation with diethylaminoethyl-dextran (30 μg/ml)(DEAE-Dextran; GIBCO) in HBSS. The infectious inoculum (ATCC VR-1310)was added to the flasks in 2 ml of serum-free media. The flasks werethen centrifuged at 1,200×g for 1 hour at 4° C. To the spun flasks wasadded 2 ml of Iscoves medium (GIBCO) containing 4 μg/ml cyclohexamide,20% FCS, 4 mM L-glutamine (Sigma), and 100 μg/ml gentamicin (Sigma). Theflasks were then incubated at 35° C. for three days at which time theinfected cells begin to lyse and release EBs. On day 4, the cultureflasks were sonicated for 20 seconds, and the cell debris removed bycentrifugation at 600×g for five minutes at room temperature. Thesupernatant containing the infectious EBs was centrifuged at 18,000×gfor 30 minutes, and the pellet resolubilized in water containing 25 mMDTT, 2% 2-mercaptoethanol, and 2% SDS.

[0165] Preparation of Purified rMOMP

[0166] Preparation of purified rMOMP was performed as follows. Thefull-length native C. pneumoniae MOMP gene was expressed in the pET-32/a(+) E. coli expression system using a polyhistidine tag (Novagen,Madison, Wis.). Briefly the MOMP was amplified under the same PCRconditions with a NcoI extension on the forward primer and a NotIextension on the reverse primer. The 5′ to 3′ sequence of the forwardprimer was AGC TTA CCA TGG TGA ATG AAA AAA CTC TTA AAG TCG GCG (SEQ IDNO: 9) and the reverse primer was ATA TGC GGC CGC TCA TTA GAA TCT GAACTG ACC AGA TAC G (SEQ ID NO: 10). The product was cut with NotI andNcoI and ligated into the multiple cloning site in a previouslylinearized pET vector. E. coli was transformed and selectively grown,lysed with a French press, and the protein extract was run over aNi-chelate column and eluted with polyhistidine. Purified rMOMP wasisolated by molecular sieve chromatography following cleavage of the tagsequence with thrombin. The purified C. pneumoniae rMOMP containing thewild-type sequence was used in the in vitro experiments.

[0167] ELISA for IL-12/p40

[0168] IL-12/p40 in culture supernatants was quantitated using sandwichELISA methodology. Antibody C17.15 was coated onto ELISA plates at 2μg/ml in 100 μl of carbonate buffer at pH 9.3. After overnightincubation at 4° C., excess antibody was washed off, and the plates wereblocked by addition of PBS containing 3% BSA. Samples and standards wereplated in triplicate and incubated overnight at 4° C. Plates were washedagain with PBS containing 0.05% Tween 20 and biotinylated anti-IL-12antibody (C15.8) antibody was added at 2 μg/ml. After one hour at roomtemperature, the plates were washed three times with PBS, andavidin-alkaline phosphatase was added followed by the addition of 1mg/ml p-nitrophenyl phosphate. The absorbance was read at 405 nm andsample concentrations of IL-12/p40 were calculated from a rIL-12/p40standard curve.

[0169] Measurement of NO Production

[0170] NO is rapidly oxidized to nitrite in culture medium.Determination of nitrite concentration, therefore, is used as ameasurement of NO production. This was done by mixing 50 μl of culturesupernatant with 50 μl of Greiss reagent (1% sulfanilamide, 0.1%naphthylethylene diamine dihydrochloride, 2.5% H₃PO₄) in individualwells of 96-well tissue culture plates (Corning). After a 10 minuteincubation at room temperature, the absorbance was read at 550 nm.Nitrite concentrations were calculated from a sodium nitrite standardcurve.

[0171] PCR for iNOS

[0172] Mouse splenic macrophages were homogenized in TRI reagent(Molecular Research Center, Cincinnati, Ohio), and total RNA wasextracted according to the manufacturer's protocol. One microgram oftotal RNA was reverse transcribed into cDNA in a 20 μl reaction mixturecontaining 50 U murine leukemia virus reverse transcriptase (MuLV RT),20 U RNase inhibitor, 1 nM of each dNTP, 2.5 μM random hexamers, 5 mMMgCl₂, and 1× buffer containing 50 mM KCl and 10 mM Tris-HCl, pH 8.3(all reagents from Perkin Elmer) using a gene amplification kit (PerkinElmer Corp., Norwalk, Conn.). PCR amplification was carried out using 5μl cDNA in a 25 μl reaction mixture containing 0.625 U AmpliTaq, 12.5pmol of each primer, 2 mM MgCl₂, and 1× buffer (Perkin Elmer). Primersused were as follows: iNOS sense: 5′-TAG AGG AAC ATC TGG CCA GG-3′ (SEQID NO: 11) iNOS antisense: 5′-TGG CAG CAT CCC CTC TGA TG-3′ (SEQ ID NO:12) GAPDH sense: 5′-TGA AGG TCG GTG TGA ACG GAT TTG GC-3′ (SEQ ID NO:13) GAPDH antisense: 5′-CAT GTA GGC CAT GAG GTC CAC CAC-3′ (SEQ ID NO:14)

[0173] The PCR reaction was carried out in a PTC-200 programmablethermocycler (M J Instruments Inc., Waltham, Mass.) for 30 cycles asfollows: iNOS, 94° C. for 30 seconds, 56° C. for 1 minute, and 74° C.for 1 minute, with a final extension for 7 minutes; GAPDH, 94° C. for 15seconds, 55° C. for 20 seconds, and 72° C. for 1 minute. Finally, 7 μlof the PCR product was run on a 1% agarose gel in TAE buffer.

[0174] B) Results

[0175] Induction of iNOS in Mouse Macrophages Exposed to C. pneumoniaeEB Antigens and Purified rMOMP

[0176] We examined the dose effect and kinetics of induction of nitritein splenic macrophage cell culture supernatants following exposure toeither EB antigens or purified rMOMP. As shown in FIGS. 8A and 8B, EBantigens of C. pneumoniae and rMOMP are each potent inducers of iNOS.Following addition of 4 μg/ml of EB antigens to macrophage cultures,nitrite levels in culture supernatants increased from 1.77±1 μM to12.7±2.4 μM. When 4 μg/ml of rMOMP was added to macrophage cultures,nitrite levels increased from 2.4±0.4 μM to 14.5±1.8 μM. Endotoxinactivity, as ascertained by limulus assay, was absent in either the EBantigen preparations or purified rMOMP, thereby excluding a possiblecontamination by LPS in these chlamydial antigen preparations as thereason for the induction of iNOS.

[0177] Increase in iNOS Activity in Cells Pre-Treated with β-IFN andCultured with EB Antigens and Purified rMOMP

[0178] Addition of β-IFN to cultures prior to the addition of either EBantigens or purified rMOMP increased iNOS activity over that induced bythe addition of either antigen alone (FIG. 9). At 48 hours, nitritelevels in supernatants increased from 5.1 μM in EB antigen-treatedcultures to 19.6 μM after prior addition of 1000 U of β-IFN (FIG. 9).Addition of β-IFN alone did not increase nitrite levels in macrophageculture supernatants.

[0179] To further establish that the increase in NO activity wasdirectly related to the addition of β-IFN, increasing concentrations ofanti-β-IFN antibody was incubated with β-IFN. Sheep anti-mouse β-IFNantibody was added to neutralize the function of β-IFN. Sheepimmunoglobulin that was added in amounts equal to that used toneutralize β-IFN was used as controls. The antigen-antibody complex wascentrifuged at 13,000×g for 30 minutes, and unbound β-IFN was removedfrom the supernatant and added to macrophages cultures along with EBantigens. As shown in FIG. 10, the addition of 10 U of β-IFN increasednitrite levels in EB antigen-treated macrophages from 8.0±0.9 μM to27.3±0.4 μM (measured at 48 hours). Incubation of β-IFN with anti-β-IFNantibody (an amount sufficient to neutralize 100 U of β-IFN) reduced theamount of nitrite in the culture supernatants to basal levels.Incubation of β-IFN with control sheep immunoglobulin did not inhibitnitrite levels, thus demonstrating the specificity of the β-IFNenhancing effect. Similar results were also obtained with addition ofβ-IFN to macrophage cell cultures prior to the addition of either rMOMPor LPS (FIGS. 11A-and 11B). Addition of 10 U of β-IFN to macrophagecultures incubated with 2 μg/ml of rMOMP resulted in a 47% increase inthe nitrite level of activity. Culture of macrophages with LPS to which10 U of β-IFN was added increased NO by 35%. Pre-incubation ofanti-β-IFN antibody abrogated the enhancement seen following addition ofβ-IFN following culture with either rMOMP or LPS.

[0180] Effect of Addition of EB Antigens and Purified rMOMP toMacrophages on iNOS Induction

[0181] We next determined if the increase in nitrite activity seenfollowing addition of EB antigens or purified rMOMP was related to anincrease in the induction of iNOS (NOS2) gene transcription. Althoughthree NOS genes are present in mammalian cells, only NOS2 is inducible.Macrophages were treated with increasing amounts of murine β-IFN in thepresence of either EB antigens or purified rMOMP. RT-PCR was performedusing NOS primers, and the strength of the PCR signal was compared withthe constitutively expressed mRNA for GAPDH. As shown in FIG. 12A,addition of β-IFN alone increased RT-PCR signals for NOS2 gene.Following addition of EB antigens (FIG. 12A) or purified rMOMP (FIG.12B), however, a further amplification of the signal was noted. Theseresults strongly suggests that both purified rMOMP and EB antigens of C.pneumoniae are capable of inducing iNOS, and this induction is enhancedfollowing pre-incubation with β-IFN.

[0182] Effect of EB Antigens and Purified rMOMP on IL-12/p40 Production

[0183] We next examined the effect of EB antigens and purified rMOMP onthe induction of IL-12/p40 in splenic macrophages. As shown in FIGS. 13Aand 13B, a dose dependent increase in IL-12/p40 is seen followingincubation with either EB antigens or purified rMOMP. Following additionof either EB antigens or purified rMOMP, a greater than 10-fold increasein IL-12/p40 was seen. Subsequent studies were done using 2 μg/ml ofantigen.

[0184] Effect of β-IFN on IL-12/p40 Production

[0185] Macrophages were pretreated with β-IFN with IL-12/p40 levels inmacrophage culture supernatants examined following addition of EBantigens of C. pneumoniae. As shown in FIG. 14, IL-12/p40 levelsdecreased from 6.6±0.2 ng/ml in EB antigen-treated cultures to 1.5±0.09in cultures treated with EB antigens for 48 hours. Following theaddition of 10 U of β-IFN, the induction of IL12/p40 is suppressed by78%. To show specificity, anti-β-IFN antibody was added to the cultures,and its effect on IL-12/p40 was examined in a manner similar to thatshown earlier. Addition of 500 U of anti-βIFN antibody abrogated theinhibitory effects of β-IFN on the induction of IL12/p40 by either EBantigens (100% reduction; FIG. 15A) or by rMOMP (54% reduction; FIG.15B). Addition of equal amounts of control sheep immunoglobulin did notaffect the inhibitory effects of β-IFN.

EXAMPLE 5 Treatment of MS by Administering Anti-Chlamydial Agents

[0186] Table 8 shows the course of therapy for a number of MS patientstreated with a combination of anti-chlamydial agent. The case historiesfor these patients are described in Table 9. Table 10 lists the standarddosages for the drugs listed in Table 8. TABLE 8 Duration Patient SexTreatment Regimen (months) Comments BL M Rifampin 2 MetronidazoleOfloxacin Metronidazole 5 Sulfamethoxazole/ Trimethoprim Levaquin — 3Discontinued therapy, had relapse Metronidazole 2 Sulfamethoxazole/Trimethoprim Levaquin Metronidazole 7 Sulfamethoxazole/ TrimethoprimLevaquin Penicillamine Rifampin 3 INH Penicillamine Probenecid MC MRifampin 9 INH Metronidazole Levaquin 6 Probably not Minocyclinecompliant — — Discontinued JM M Metronidazole 7 OfloxacinSulfamethoxazole/ Trimethoprim Minocycline Amoxicillin 4 LevaquinSulfamethoxazole/ Trimethoprim Amoxicillin 3 Levaquin Sulfamethoxazole/Trimethoprim Probenecid LL F Metronidazole 15 Levaquin MinocyclinePenicillamine 1 Levaquin Minocycline Probenecid FO M Prednizone 0.25Phased in over several days to mitigate effect of therapy Metronidazole2 Clarithromycin Clarithromycin 1 Stopped metronidazole due topersistence of side effects Clarithromycin 0.5 Kemet Metronidazole 6Began phasing Clarithromycin metronidazole back in Kemet over a monthMetronidazole 1 Began two week Clarithromycin switchover to KemetAmoxicillin Amoxicillin Metronidazole 2 Clarithromycin AmoxicillinMetronidazole 6 Clarithromycin Amoxicillin Probenecid JC F Amoxicillin 1Amoxicillin 1 Probenecid Amoxicillin 1 Probenecid Sulfamethoxazole/Trimethoprim Amoxicillin 7 Probenecid Sulfamethoxazole/ Trimethoprim INHFW M Penicillamine 7 Metronidazole Doxycycline Penicillamine 5 INHSulfamethoxazole/ Trimethoprim Probenecid — —

[0187] TABLE 9 Patient Case History BL First symptoms began withnumbness of the left arm and leg which rapidly progressed to a partialBrown-Sequard syndrome (i.e. cord myelitis) with an associated urinaryretention. Despite therapy with corticosteroids, and β-IFN, he rapidlyprogressed over the next three months with an EDSS = 8.0 (triplegic plusspeech and swallowing impairments). A positive CSF PCR and culture forC. pneumoniae led to treatment with combination antibiotics. The patientimproved in all aspects of neurologic function over the following sixmonths. His EDSS score nine months later was 3.0 with return to work androutine athletic activities. His neurological status remains stable andhe continues on an anti-chlamydial combination regimen. MC This patienthad a ten year history of MS with evidence of progressive ataxia andweakness in the legs. Over five months his EDSS score worsened from a7.0 to 8.0. His CFS was positive by PCR for C. pneumoniae and he wasplaced on combination antibiotics. Over the next six months he graduallyimproved in his balance, coordination and lower extremity strength. Hismost recent EDSS score was 6.5. JM Initially seen with rapidlyprogressive paraparesis secondary to MS. He failed to response tocorticosteroids on two successive occasions. Five months later, his EDSSscore was 7.5. Following a positive C. pneumoniae PCR, he was placed oncombination antibiotics. He has gradually gain strength in his lowerextremities and five months later was able to walk with a walker (EDSS =6.5) while maintaining on combination antibiotics. LL Patient with along history (14 years) of secondary progressive MS with recentprogressive bulbar symptoms, axtaxia, and paraplegia (EDSS = 8.5). PCRfor the MOMP gene of C. pneumoniae in the CSF was positive. She wasplaced on combination antibiotics with no further progression ofsymptoms for the last six months. AN Long history of MS and wheel chairbound for approximately ten years. She has received continuous physicaltherapy to retain leg muscle tone. Following approximately six months ofcombination antibiotics, she was able to stand unaided and take severalunaided steps. She reports significant decrease in fatigue and cognitivedysfunction. She remains on combination antibiotics and other supportivemedications. FO Wheel chair bound with a long history of MS with atwo-three year progression of severe dysarthriae and incontinence. Oncombination antibiotics (14 months) he has had improvement of speech andincontinence. Speech, ability to open mouth for dentist, stamina allimproved. He can stand better on his own mid-transfer, but remainswheelchair-bound. JC Diagnosis of MS with development of a foot dropapproximately one year prior to therapy requiring the use of a cane inwalking. Approximately four months after initiation of combinationantibiotic therapy, patient reports reversal of foot drop and no longerrequires a cane. She continues on antibiotic therapy. FW Male with a 15year history of MS. Used a cane for a rolling, unstable gait. Easilyfatigued. After 12 months of combination antibiotics, was able to walkwithout cane or excessive fatigue, although his gait can still wander.Can easily make it across the parking lot, which had previously been achallenge. Stopped antibiotics even though was still PCR positive; plansto restart therapy if he has another flare- up.

[0188] TABLE 10 Drug Generic Unit dosage Daily dosage CupraminePenicillamine 250 mg 2X Amoxicillin 500 mg 2X Flagyl Metronidazole 500mg 2X INH 300 mg 1X Rifampin 300 mg 2X Floxin Ofloxacin 400 mg 2XLevaquin 500 mg 1X Bactrim SMZ/TMP Double Strength 2X BiaxinClarythromycin 500 mg 2X Minocycline 100 mg 2X Doxycycline 100 mg 2XProbenecid 500 mg 2X

[0189] The efficacy of long-term administration of combination therapyin the treatment of 11 patients with secondary progressive MS and onepatient with primary progressive MS (patient #6) is shown in Table 11.All 12 patients were positive by PCR for the MOMP gene of C. pneumoniaein the CSF. In 10 of 12 patients, the highest Expanded Disability StatusScale (EDSS; Kurtzke, Neurology 33:1444-1152, 1983) score reached wassustained for six months. In patient #1, the maximal EDSS was presentfor four months and improved when he was treated with antibiotics forurosepsis. The antibiotic regimen in eight of 12 patients was acombination of rifampin (300 mg twice daily), amoxicillin (500 mg twicedaily) and probenecid (500 mg daily). Patient #5 discontinued use ofamoxicillin after three months and was continued on rifampin alone.Patients #3 and #10 were administered rifampin and levofloxacin. Inpatient #12, azithromycin was substituted for rifampin in view of thegastrointestinal side effects. Patients #1, #10, #11, and #12 alsoreceived β-IFN.

[0190] Overall, six patients improved by the EDSS and, in each case,improvement has been maintained for at least six months. Of the sixpatients who showed no changes on the EDSS, patient #2 had sustainedimprovement in upper extremity function as measured by the nine hole pegtest. In patient #4, the EDSS did not change, but her ambulation index(time to walk 25 feet) improved from 52.8 seconds at the time ofinstitution of antibiotics to 32.5 seconds at time of completion. Inpatient #8, there was a sustained improvement in his visual acuity.Patient #10 had an increase in her EDSS from 6.0 to 8.0 while onrifampin and levofloxacin. TABLE 11 Other Duration of PCR Signal PatientAge/Sex EDSS Duration Antibiotics Steroids Drugs Antibiotics EDSS IIImproved Post-antibiotics 1 34/M 7.5 3 m Rifampin No β-IFN1b 12 m 6.5Yes Absent Amoxicillin 2 36/F 8.5 6 m Rifampin No No 12 m 8.5 YesDecreased Amoxicillin 3 49/F 8.5 9 m Rifampin No No 15 m 8.0 Yes Nochange Levoflaxacin 4 41/F 6.5 18 m Rifampin No No 9 m 6.5 Yes NDAmoxicillin 5 54/M 8.0 9 m Rifampin No No 10 m 7.5 Yes ND 6 33/F 8.5 5 mRifampin Yes No 12 m 8.0 Yes Absent Amoxicillin 7 57/M 8.5 24 m RifampinNo No 12 m 8.5 No ND Amoxicillin 8 34/M 3.0 8 m Rifampin Yes Cop-1 12 m2.5 Yes ND Amoxicillin 9 38/F 6.5 6 m Rifampin No No 9 m 5.5 YesDecrease Amoxicillin 10 25/F 6.0 3 m Rifampin Yes β-IFN 12 m 8.0 No NDLevoflaxacin 11 26/F 6.0 6 m Rifampin No β1a 6 m 3.0 Yes ND Amoxicillin12 42/F 6.0 6 m Azithromycin Yes β-IFN1a 9 m 6.0 No Absent

EXAMPLE 6 Animal Models for the Identification of Drugs for theTreatment of MS

[0191] The discovery that chlamydial infection correlates with MS allowsfor the development of animal models for drug identification. Forexample, an animal (e.g., a mouse, rat, or rabbit) can be infected byinjecting Chlamydia into the ventricles of the brain. Once infection ofthe brain has been established, candidate compounds can be administeredto the animal using any mode of administration described above for theadministration of compounds to humans. The ability of the compound toeradicate the infection can be ascertained by performing one or more ofthe assays described herein. For example, the detection of chlamydialDNA (e.g., the MOMP gene or the 16S RNA gene) in the CSF or blood of theanimals can be performed using the methods described in Examples 1 and2. If desired, the animal can be sacrificed and the tissue examinedusing standard histological methods for the detection of chlamydialinfection.

[0192] Alternatively, MS therapeutics may be identified using any othermethod for identifying anti-chlamydial agents. A number of suchscreening assays are described in co-pending U.S. patent applicationSer. No. 09/073,661.

[0193] Other Embodiments

[0194] All patent applications and publications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent patent application and publication was specificallyand individually indicated to be incorporated by reference.

[0195] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications. This application is intended to cover anyvariations, uses, or adaptations following, in general, the principlesof the invention and including such departures from the presentdisclosure within known or customary practice within the art to whichthe invention pertains and may be applied to the essential featureshereinbefore set forth.

[0196] Other embodiments are within the claims.

1 14 1 38 DNA Artificial Sequence Based on Chlamydia pneumoniae 1atgaaaaaac tcttaaagtc ggcgttatta tccgccgc 38 2 40 DNA ArtificialSequence Based on Chlamydia pneumoniae 2 ttagaatctg aactgaccagatacgtgagc agctctctcg 40 3 24 DNA Artificial Sequence Based on Chlamydiapneumoniae 3 gctgctgcaa actatactac tgcc 24 4 25 DNA Artificial SequenceBased on Chlamydia pneumoniae 4 gaatcagtag tagacaatgc tgtgg 25 5 22 DNAArtificial Sequence Based on Chlamydia pneumoniae 5 tttagtggcggaagggttag ta 22 6 22 DNA Artificial Sequence Based on Chlamydiapneumoniae 6 cacatatcta cgcatttcac cg 22 7 25 DNA Artificial SequenceBased on Chlamydia pneumoniae 7 ctttcggttg aggaagagtt tatgc 25 8 29 DNAArtificial Sequence Based on Chlamydia pneumoniae 8 tcctctagaaagatagtttt aaatgctga 29 9 39 DNA Artificial Sequence Based on Chlamydiapneumoniae 9 agcttaccat ggtgaatgaa aaaactctta aagtcggcg 39 10 40 DNAArtificial Sequence Based on Chlamydia pneumoniae 10 atatgcggccgctcattaga atctgaactg accagatacg 40 11 20 DNA Artificial Sequence Basedon Chlamydia pneumoniae 11 tagaggaaca tctggccagg 20 12 20 DNA ArtificialSequence Based on Chlamydia pneumoniae 12 tggcagcatc ccctctgatg 20 13 26DNA Artificial Sequence Based on Chlamydia pneumoniae 13 tgaaggtcggtgtgaacgga tttggc 26 14 24 DNA Artificial Sequence Based on Chlamydiapneumoniae 14 catgtaggcc atgaggtcca ccac 24

What is claimed is:
 1. A method of treating Multiple Sclerosis in apatient diagnosed to have multiple sclerosis, said method comprisingadministering a rifamycin to the individual in an amount effective totreat said multiple sclerosis.
 2. The method of claim 1, furthercomprising administering to the patient a compound selected from thegroup consisting of an azalide, macrolide, ketolide, and streptogramin.3. The method of claim 1, further comprising administering to thepatient ampicillin or amoxicillin; and probenecid.
 4. The method ofclaim 1, further comprising administering a nitroimidazole to thepatient.
 5. The method of claim 1, further comprising administering aquinolone or fluoroquinolone to the patient.
 6. The method of claim 1,further comprising administering a sulfonamide and an isonitotiniccongener to the patient.
 7. The method of claim 1, further comprisingadministering a tetracycline to the patient.
 8. The method of claim 1,wherein the individual is also administered an effective amount of acompound that increases iNOS activity.
 9. The method of claim 8, whereinthe agent that increases iNOS activity is a type-1 interferon, asynthetic type-1 interferon analog, or a hybrid type-1 interferon,wherein the type-1 interferon analog or hybrid binds to the samereceptor as a naturally-occurring type-1 interferon.
 10. The method ofclaim 9, wherein the type-1 interferon is β-interferon.
 11. The methodof claim 1, wherein the administering is continued until the individualtests negative for elementary body phase Chlamydia, replicating phaseChlamydia, and cryptic phase Chlamydia.
 12. The method of claim 11,wherein said Chlamydia is Chlamydia pneumoniae.
 13. The method of claim1, wherein the administration is continued for at least 45 days.
 14. Themethod of claim 13, wherein the administration is continued for at least90 days.
 15. The method of claim 14, wherein the administration iscontinued for at least 180 days.